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SELECTED ARTICLES MARCH 2015 www.jem.org

T CELLS
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 Welcome
T Cells
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 the regulation and
effector function of T cells.
Our collection begins with an insight from Luc Van Kaer describing the findings from Bougeois
et al. establishing a role for bee and wasp venom enzymes in generating neoantigens that activate
CD1a-restricted T cells.They show that phospholipase A2, which is present in insect venom, acts on
human skin-derived phospholipids to release fatty acid antigens that are then loaded onto CD1a and
presented to T cells, which subsequently become activated and produce interleukin (IL)-22.
Mutations in dedicator of cytokinesis 8 (DOCK8) can result in an inherited combined immuno
deficiency in humans. Zang et al. demonstrate that susceptibility to skin infections seen in patients with
DOCK8 mutations is due to a form of catastrophic cell death the authors term cytothripsis (cell shattering),
as T cell traffic through the collagen-dense tissue network of the skin. This impaired flexibility prevents the
generation of skin-resident memory CD8+ T cells that are needed to control herpesvirus skin infections. An insight by Stuart Tangye
accompanies the article and speculates on how the findings could be used to limit pathology incurred by skin infections.
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 highlight 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 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. IFNg
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 (Treg) can modulate CD8+
T cell exhaustion during chronic lymphocytic choriomeningitis virus infection in mice. Their findings show that depletion of
Tregs can expand functional virus-specific CD8+T cells, rescuing exhausted CD8+ T cell subpopulations. Treg 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
Treg depletion that is able to reduce viral load. These findings suggest that Tregs have the ability to contribute to the maintenance
of exhausted CD8+ T cells during chronic infection.
Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through thymic expression of tissue-specific antigens
(TSAs). Modulating central tolerance is an attractive therapeutic strategy for cancer treatment. Khan et al. demonstrate that in vivo
blockade of RANKL can inhibit the development and turnover of mTECs and alter central T cell tolerance. In vivo RANKL
blockade in mice transiently depletes Aire and TSA expression in the thymus inhibiting negative selection. RANKL blockade
can also rescue melanoma-specific T cells from thymic deletion, with tumor-specific effector CD4+ T cells facilitating host survival
in response to tumor challenge.
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type1
(HTLV-1). Increased expression of CCR4 is a hallmark of ATLL, but it is not clear whether dysregulated CCR4 contributes to
disease pathogenesis. An article by Nakagawa et al. identifies recurring somatic mutations of CCR4 in ATLL patients, finding
that a CCR4 gain-of-function mutation impairs CCR4 internalization and increases cell migration and chemotaxis in vitro. An
accompanying insight by Kevin Shannon discusses the implications of CCR4 mutations, PI-3K signaling downstream of CCR4,
and the translational potential of CCR4 blockade.
Acute cerebral ischemia reperfusion injury is mediated in part by T cells. Clarkson et al. show that brain-infiltrating
CD4+T cells sustain neuroinflammation after stroke in mice by producing IL-21 and increasing neuronal death. Treatment with
an IL-21 decoy receptor or genetic lack of IL-21 protected animals from brain injury following stroke, offering a potential
target for immunotherapy. Additionally, analysis of postmortem human brain tissue confirmed that IL-21 localizes to CD4+T cells
surrounding acute stroke lesions.
Together these studies provide new insights into the biology and mechanisms of T cell responses in several disease and
pathogen conditions and offer insight into therapeutics. We hope you enjoy this complimentary copy of our T cell collection.
We invite you to explore additional collections at www.jem.org and to follow JEM on Facebook, Google+, and Twitter.
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 Selected Articles March 2015

Bee venom stirs up buzz in antigen presentation

Luc Van Kaer

Bee venom processes human skin lipids for presentation by CD1a

Elvire A. Bourgeois, Sumithra Subramaniam, Tan-Yun Cheng, Annemieke De Jong, Emilie Layre, Dalam Ly,
Maryam Salimi, Annaliza Legaspi, Robert L. Modlin, Mariolina Salio, Vincenzo Cerundolo, D. Branch Moody,
and Graham Ogg

T cells require DOCK8 for flexibility and function

Stuart Tangye

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity

Qian Zhang, Christopher G. Dove, Jyh Liang Hor, Heardley M. Murdock, Dara M. Strauss-Albee, Jordan A. Garcia,
Judith N. Mandl, Rachael A. Grodick, Huie Jing, Devon B. Chandler-Brown, Timothy E. Lenardo, Greg Crawford,
Helen F. Matthews, Alexandra F. Freeman, Richard J. Cornall, Ronald N. Germain, Scott N. Mueller, and Helen C. Su

FLUshing in the bathroom

Carolina Amezcua, Nicola Gagliani, and Richard Flavell

Respiratory influenza virus infection induces intestinal immune injury via microbiotamediated
Th17 celldependent inflammation
Jian Wang, Fengqi Li, Haiming Wei, Zhe-Xiong Lian, Rui Sun, and Zhigang Tian

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

CCR4 drives ATLL jail break

Kevin Shannon

Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma

Masao Nakagawa, Roland Schmitz, Wenming Xiao, Carolyn K. Goldman, Weihong Xu, Yandan Yang, Xin Yu,
Thomas A. Waldmann, and Louis M. Staudt

Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance

Imran S. Khan, Maria L. Mouchess, Meng-Lei Zhu, Bridget Conley, Kayla J. Fasano, Yafei Hou, Lawrence Fong,
Maureen A. Su, and Mark S. Anderson

T cellderived interleukin (IL)-21 promotes brain injury following stroke in mice

Benjamin D.S. Clarkson, Changying Ling, Yejie Shi, Melissa G. Harris, Aditya Rayasam, Dandan Sun, M. Shahriar Salamat,
Vijay Kuchroo, John D. Lambris, Matyas Sandor, and Zsuzsanna Fabry
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INSIGHTS

Bee venom stirs up buzz in antigen presentation

While most T cells recognize peptide antigens presented by major histocompatibility complex (MHC) class I
and class II molecules, some T cells react with lipid antigens presented by MHC-related CD1 proteins. In this
issue, Bourgeois et al. establish the concept that enzymes in bee venom can cleave host skin-derived phospho-
lipids into lipid neoantigens that activate CD1a-restricted T cells to promote a local inflammatory response.
Humans have four CD1 proteins: CD1a, CD1b, CD1c, and CD1d. The CD1a molecule has long been
known as a specific surface marker for human Langerhans cells, and recent studies have shown that CD1a-
restricted T cells are recruited to the skin. Such T cells are intrinsically autoreactive, recognizing skin-specific
oils produced by sebaceous glands. Insight from Luc
In searching for potential contributions of CD1a-reactive T cells to inflammatory responses in the Van Kaer
skin, Bourgeois et al. investigated human T cell responses to the venoms of bees and wasps. They found
that such venoms induce a CD1a-mediated response in both individual T cell clones and primary human T cells. However,
further analyses revealed that the antigenic substance unex-
pectedly partitioned within the lipid rather than the protein
fractions, raising the possibility that enzymes in the venom
generate lipid antigens by cleaving common phospholipids.
The investigators were able to zoom in on phospholipase
A2 (PLA2), an enzyme that is abundant in insect and snake
venoms, which acts on phospholipids to release fatty acids
and lysophospholipids.
These intriguing findings support a model of T cell acti-
vation involving insect-mediated PLA2 introduction into the
skin, inducing the release of fatty acid neoantigens from com-
mon skin phospholipids, subsequently sampled by Langerhans
cells, loaded onto CD1a molecules, and presented to T cells.
Activated CD1a-reactive T cells produce IL-22, a cytokine
that plays a role in antimicrobial defenses, promotes keratino-
cyte proliferation, and contributes to the pathogenesis of a
variety of skin inflammatory diseases. In this manner, CD1a-
reactive T cells may contribute to the local inflammatory
response to venoms. The findings also provide a potential
molecular explanation for the generation of autoantigens in
normal skin, which expresses secreted phospholipases. It has
been proposed that such natural lipids are present at the surface
Phospholipase A2 (PLA2) in bee venom is introduced into human skin
and cleaves ubiquitous phospholipids into free fatty acids and lyso-
of the skin and are thus inaccessible to CD1a-restricted T cells
phospholipids that function as neoantigens. These fatty acid neoan- in the dermis. However, a breach in the skin barrier might
tigens are now available for sampling by epidermal Langerhans cells, make these natural oils available to Langerhans cells in the epi-
which load them onto CD1a proteins and present them at their surface dermis, resulting in their presentation to CD1a-reactive T cells
to skin-resident CD1a-reactive T cells. These T cells produce cytokines and the subsequent induction of antimicrobial and inflam-
such as IL-22 that contribute to the inflammatory response. matory responses. Moreover, infections and inflammatory
processes can modulate the expression patterns of phospho-
lipases in the skin, which may represent a mechanism to control inflammation via CD1a-reactive T cells. Finally, certain skin
pathogens secrete PLA2 enzymes, raising the possibility that such infections can induce CD1a-mediated T cell responses that
contribute to host immunity and disease pathogenesis. These proposed scenarios, together with their potential therapeutic
applications, will provide rich and fertile avenues for future investigation.
Bourgeois, E.A., et al. 2015. J. Exp. Med. http://dx.doi.org/10.1084/jem.20141505.

Luc Van Kaer, Vanderbilt University School of Medicine: luc.van.kaer@vanderbilt.edu


 Article

Bee venom processes human skin lipids

for presentation by CD1a
Elvire A. Bourgeois,1* Sumithra Subramaniam,2,3* Tan-Yun Cheng,1
Annemieke De Jong,1 Emilie Layre,1 Dalam Ly,1 Maryam Salimi,2,3
Annaliza Legaspi,4,5 Robert L. Modlin,4,5 Mariolina Salio,2,3
Vincenzo Cerundolo,2,3 D. Branch Moody,1** and Graham Ogg2,3**
1Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Womens Hospital, Harvard

Medical School, Boston, Massachusetts, 02114

2MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine and 3University of Oxford NIHR Biomedical

Research Centre, Oxford, Oxfordshire OX3 9DS, England, UK

4Division of Dermatology, David Geffen School of Medicine, 5Department of Microbiology, Immunology and Molecular

Genetics, University of California Los Angeles, Los Angeles, CA 90095

Venoms frequently co-opt host immune responses, so study of their mode of action can
provide insight into novel inflammatory pathways. Using bee and wasp venom responses as
a model system, we investigated whether venoms contain CD1-presented antigens. Here, we
show that venoms activate human T cells via CD1a proteins. Whereas CD1 proteins typically
present lipids, chromatographic separation of venoms unexpectedly showed that stimula-
tory factors partition into protein-containing fractions. This finding was explained by
demonstrating that bee venomderived phospholipase A2 (PLA2) activates T cells through
generation of small neoantigens, such as free fatty acids and lysophospholipids, from
common phosphodiacylglycerides. Patient studies showed that injected PLA2 generates
lysophospholipids within human skin in vivo, and polyclonal T cell responses are dependent
on CD1a protein and PLA2. These findings support a previously unknown skin immune
response based on T cell recognition of CD1a proteins and lipid neoantigen generated
in vivo by phospholipases. The findings have implications for skin barrier sensing by T cells
and mechanisms underlying phospholipase-dependent inflammatory skin disease.

CORRESPONDENCE
D. Branch Moody:
bmoody@partners.org
OR
Graham Ogg:
graham.ogg@ndm.ox.ac.uk
Abbreviations used: APC, anti-
gen presenting cells; DDM,
dideoxymycobactin; LC,
Langerhans cells; mDC,
monocyte-derived DC; PC,
phosphatidylcholin; PLA2,
phospholipase A2.
Extensive evidence for the important role of
peptideMHC complexes in T cell activation
evolved into a widespread belief that peptides are
the only common and natural target of human
T cell responses. Therefore, until recently, nearly
all human clinical studies of T cell action in au-
toimmune, allergic, and infectious diseases were
targeted at peptide antigens. For example, most
candidate antigens for human T cellmediated
autoimmune diseases are proteins (Klein et al.,
2014). Subunit vaccines (Tameris et al., 2013)
and diagnostic tests (Lalvani and Pareek, 2010)
rely on defined peptide motifs, and mouse models
*E.A. Bourgeois and S. Subramaniam contributed equally to
this paper.
** D.B. moody and G. Ogg contributed equally to
this paper.
A. De Jongs present address is Columbia University, Depart-
ment of Dermatology, New York, NY 10032.
D. Lys present address is University Health Network, Uni-
versity of Toronto, Department of Immunology, Toronto,
Ontario M5G 1L7, Canada.
of autoimmunity start with protein and peptide
antigen vaccination. However, the discovery of
the function of CD1a, CD1b, CD1c, and CD1d
proteins (McMichael et al., 1979; Calabi and
Milstein, 1986) as antigen-presenting molecules
expands the biochemical spectrum of natural
antigens for T cells to include many types of lip-
ids (Porcelli et al., 1989, 1992; Kronenberg and
Kinjo, 2005).
CD1 proteins are conserved among mam-
mals (Kasmar et al., 2009) and are expressed at
high density on thymocytes and professional
APCs in the periphery, including Langerhans
cells (LCs), B cells, macrophages and myeloid
DCs (Dougan et al., 2007). In cells, CD1 pro-
teins bind and display hundreds of molecular
species of self-sphingolipids, phospholipids, and
2015 Bourgeois et al. This article is distributed under the terms of an
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J. Exp. Med. 2015 Vol. 212 No. 2 149163 149
www.jem.org/cgi/doi/10.1084/jem.20141505
acylglycerides (Huang et al., 2011), and >20 types of stimula-
tory lipid antigens for T cells are now known (Young and
Moody, 2006). The molecular bases by which lipids are rec-
ognized by T cells are well established through crystal struc-
tures of CD1, CD1 bound to lipid, and CD1-lipid bound to
a TCR (Zeng et al., 1997; Gadola et al., 2002; Borg et al.,
2007). The alkyl chains of lipids are sequestered within the
grooves of CD1 proteins, allowing the carbohydrate, sulfate,
phosphate, and other polar elements to protrude and interact
with TCRs.
Despite the wealth of molecular and cell biological data
showing that mammalian T cells can recognize lipids,
translational research to determine the roles of CD1-restricted
lipid antigens in vivo or during diseases that are commonly
seen by physicians is limited. Most reported studies have focused
on CD1d and CD1d-restricted T cells, known as NKT cells,
because CD1d proteins are the only CD1 isoform expressed in
commonly used mouse models (Godfrey and Rossjohn, 2011).
Yet CD1a, CD1b, and CD1c differ from CD1d proteins, and
from one another, in their trafficking and tissue distribution,
suggesting that they exert different physiological roles (Kasmar
et al., 2009). Notably, CD1a, unlike CD1b and CD1c pro-
teins, has been known for decades as a phenotype-specific
marker of human epidermal LCs (Dougan et al., 2007). In
addition to studies of guinea pigs (Hiromatsu et al., 2002a,b)
and transgenic mice (Felio et al., 2009), the functions of
CD1a, CD1b, and CD1c proteins have been studied in humans
during tuberculosis (Moody et al., 2000) and seasonal allergy
(Agea et al., 2005), and as in other pathogens that infect humans
(Zeissig et al., 2010). However, human studies rely mostly on
T cell clones whose functions change over time during in vitro
culture and may not represent the natural populations of T cells
in vivo.
To study polyclonal autoreactive human T cells ex vivo,
APCs that lack detectable surface expression of MHC proteins
(K562 cells) were engineered to express CD1a, CD1b, CD1c,
or CD1d proteins at high density (K562-CD1). K562-CD1
cells provide lipid autoantigens, and such MHClow CD1high
APCs largely avoid MHC alloreactivity (de Jong et al., 2010).
Therefore, T cells from groups of randomly chosen or un-
related donors can be tested for antigen responses, allowing
cohort studies and quantitation of CD1 autoreactivity in
healthy subjects. Three recent studies show that CD1a auto-
reactive T cells are present in the peripheral blood of nearly
all humans tested, defining CD1a autoreactive T cells as a dis-
tinct component of the human immune system (de Jong et al.,
2010, 2014; de Lalla et al., 2011). These studies also found
that the rates of T cell autoreactivity are higher to CD1a, as
compared with CD1b, CD1c and CD1d. In addition to stud-
ies of peripheral blood, recent studies implicate CD1a auto-
reactive T cells as functioning in organ-specific immunity in
the skin. CD1a is the major human CD1 protein expressed at
high density on LCs (Dougan et al., 2007), and CD1a auto-
reactive T cells express skin-specific homing receptors (cuta-
neous lymphocyte antigen, CCR4, CCR6, and CCR10)
and home to the skin in large numbers (de Jong et al., 2010).
150
In addition, skin-specific oils, including squalene, function as
autoantigens presented by CD1a proteins (de Jong et al., 2014).
Venoms act within the skin to cause pain or death by
transfer to highly unrelated organisms; they involve multiple
active substances that typically function by coopting host
responses and thus have a long history as useful probes of nat-
ural host inflammatory responses. In particular, venom-induced
immune responses have been relevant beyond venom re
activity and have provided broader immunological insights
in to novel pathways of inflammation and tolerance (Bil and
Bonifazi, 2008; Aslam et al., 2010; Gutierrez and Rodewald,
2013). Because bee and wasp venom responses are localized
initially in skin, where CD1a is so abundantly expressed, and
because the lipidic content of the bee and wasp venoms was
unexplored, we sought to use bee and wasp venom as a model
system to investigate novel pathways of inflammation in the
skin. After initial studies documented a CD1a-mediated re-
sponse in individual T cell clones and among cohorts of pa-
tients, mechanistic studies tracked the antigenic substance to
venom proteins. By investigating venom-derived proteins,
we discovered an unexpected mechanism by which venom
phospholipases create lipid neoantigens in vivo providing a
new view of venom responses and insight into the role of
phospholipases in CD1a biology.
RESULTS
Responses of T cells to CD1 molecules and venom
Stinging insects in the genera vespula (wasp) and apis (bee)
generate a local intradermal inflammatory response. To in-
vestigate a possible role of CD1, we obtained CD3+ T cells
from healthy adult individuals and mixed them with wasp
venom-treated and irradiated K562-CD1 APCs, which were
mock transfected or transfected with either CD1a, CD1b,
CD1c, or CD1d. As expected based on prior work (de Jong
et al., 2014), we observed low but detectable alloreactive back-
ground responses of 1 in 2,000 T cells after 1214 d in cul-
ture to mock-transfected target cells in donor C1175 (Fig. 1 A,
left), which is representative of three donors. In the absence
of venom, T cells showed responses to K562-CD1a cells at
rates higher than to mock transfected cells or cells expressing
other CD1 isoforms, confirming the presence and relatively
high rates of CD1a autoreactive T cells seen previously (de Jong
et al., 2010, 2014; de Lalla et al., 2011). Further, polyclonal re-
sponses to wasp venom above background levels were detected,
but were seen only when using CD1a-expressing cells as tar-
gets (P < 0.05, Fig. 1 A, left, representative of three donors).
These responses demonstrate that a T cell response to wasp
venom is restricted by CD1a proteins.
Apis mellifera (honeybee or bee) venom also causes skin
immune responses and shares certain mechanisms of immune
action with wasp venom (Mller et al., 2009). For example,
patients that develop hypersensitivity toward wasp venom can
also show reactivity to bee venom (Egner et al., 1998).Therefore,
we next investigated the CD1-restricted T cell response to
bee venom and found a pattern of response that was similar to
that of wasp venom, including augmentation of the baseline
Bee venom generates CD1a ligands | Bourgeois et al.
 Ar ticle

Figure 1. Vespula spp and Apis mellifera venoms induce a preferential activation of CD1a-restricted T cells among the group1-CD1
reactive cells. (A) T cells were isolated by CD3 MACS beads from healthy donor PBMCs and cultured for 1214 d with IL-2 and irradiated K562
cells transfected with CD1a (K562-CD1a), CD1b (K562-CD1b), CD1c (K562-CD1c), CD1d (K562-CD1d), or an empty vector (K562) in the presence
of venom. CD1 reactivity was then examined by IFN- ELISpot with transfected or untransfected K562 cells either in the presence or absence of
Vespula spp or Apis mellifera venoms. Representative data for one donor (C1175) of three are shown. (B) The CD1a-restricted T cell response in
the presence or absence of anti-CD1a (donor C1098) and Vespula spp and Apis mellifera venoms. CD1a-restricted, venom-specific responses were
measured in 21 donors for Vespula spp venom (C) and Apis mellifera venom (D). mDC or in vitro LClike cells derived from CD14+ cells were pulsed
with 1 g/ml wasp venom (E) or bee venom (F) and incubated with the T cells in the presence or absence of anti-CD1a antibody or isotype control.
IFN- production was measured by IFN- ELISpot. Representative data for one donor (C556 [E] and C560 [F]) of three are shown. Data were mean
of triplicate measurements SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

JEM Vol. 212, No. 2 151

autoreactive response by venom in the presence of CD1a
(Fig. 1 A, right). Thus, this pilot study provided proof of prin
ciple for CD1a-mediated human cellular responses to wasp
and bee venom among human polyclonal T cells.
The total IFN- response of fresh polyclonal T cells con-
sisted of three identifiable components: a response to K562,
an increment in response to CD1a and a further increment to
added venom, consistent with CD1a presentation of venom
antigens. Expansion of T cells with antigen ex vivo offered
the opportunity to more definitively establish the molecular
mechanisms involved. We cultured T cells in IL-2 for 2 wk
in the presence of wasp venomtreated K562-CD1a cells to
permit the outgrowth of responder cells. ELISpot studies
clearly confirmed reactivity to CD1a alone, as well as to CD1a
plus wasp venom, but no response to venom was seen in the
absence of CD1a (Fig. 1 B, left, donor C1098). Further, anti-
CD1a mAb blocked the response to background levels (P <
0.05), proving that CD1a is required. A similar pattern of re-
sponse was seen to T cells treated with K562-CD1a and bee
venom (Fig. 1 B, right).
CD1a-restricted venom responses in a healthy cohort
Next, we sought to test this pattern of wasp venom responses
in a larger cohort of 21 healthy individuals. Measuring mean
IFN- spots for all donors, we observed higher (P < 0.0001)
response to CD1a transfectants, as compared with untrans-
fected target cells (Fig. 1 C). Similar to the pilot studies, we
detected higher spot numbers when wasp venom was added,
but only when CD1a was present (P < 0.001). We observed
a pattern of IFN- production to bee venom that was depen-
dent on CD1a expression (P < 0.001) and similar in magni-
tude to that seen with wasp venom (Fig. 1 D).
K562 cells are transformed cells that represent useful tools
which bypass MHC alloreactivity. The two types of primary
human skin APCs that express CD1a proteins are myeloid
DCs and LCs. To investigate whether these cell types mediate
the venom response similarly to K562 cells, we differentiated
monocytes with GM-CSF and IL-4 to produce monocyte-
derived DCs (mDCs), which mimic key features of myeloid
DCs. In addition we activated cells in vitro with cytokines to
mimic LCs (in vitro LCs), including high CD1a expression
(Porcelli et al., 1992; Sallusto and Lanzavecchia, 1994; Caux
et al., 1997; Geissmann et al., 1998). Both types of human
primary APCs were able to mediate wasp venom (Fig. 1 E)
and bee venom (Fig. 1 F) responses, which were abrogated in
the presence of anti-CD1a. Overall, this study showed that
CD1a autoreactivity and CD1a-dependent venom reactivity
are common in humans. This pattern of combined CD1a
autoreactivity and antigen-dependent reactivity is reminiscent
of invariant NKT cells, which are activated weakly by CD1d
and strongly by CD1d with lipid antigen (Kawano et al., 1997).
Isolation of stimulatory factors from venom
To neutralize diverse predators or prey, natural venoms are
usually high potency toxins that have multiple mechanisms of
action (King et al., 2003). Repeated exposure of wasp and
152
bee venoms to lymphocytes resulted in apparent toxicity and
cell death based on monitoring of T cells by Trypan blue
exclusion. We could not generate long-term T cell lines by
direct and repeated exposure to venom in vitro. Instead, we
screened existing panels of CD1a-reactive T cell clones for
responses to Vespula species (wasp) and Apis mellifera (bee)
venoms. To minimize toxicity and interrogate the mecha-
nisms of venom action, we switched from K562-CD1a acti-
vation assays (cellular assays) and took advantage of assays in
which biotinylated CD1a proteins are coated onto streptavi-
din plates (plate assays). In the plate assay, CD1a proteins are
pulsed with venom-derived products, washed and then ex-
posed to T cells. This method diminishes venom exposure
to APCs and responding cells, thereby minimizing toxicity.
Also, the plate method might favor detection of stimulants
that act by forming complexes with CD1 proteins in prefer-
ence to indirect effects of venom on APCs or T cells that do
not involve binding to CD1 proteins. The T cell clone BC2
showed a dose-dependent increase in IFN- secretion when
cultured in the presence of CD1a proteins pretreated with
wasp venom (Fig. 2 A). The response was likely to be specific
antigen recognition rather than a general mitogenic activa-
tion, because no response was seen when plates were coated
with CD1b, CD1c (Fig. 2 A), or CD1d (not depicted), or
when venom-treated CD1 proteins were exposed to another
T cell line, CD8-2, which normally recognizes dideoxymy-
cobactin lipopeptides presented by CD1a (Moody et al.,
2004; Fig. 2 B). Furthermore, we observed a similar dose-
dependent recognition by T cell clone BC2 of bee venom in
the presence of CD1a proteins (Fig. 2 C).
Using cellular K562 assays, we again observed a CD1-
dependent and dose-dependent response to bee venom, but
this time with significant CD1a autoreactivity. The higher
responses seen in cellular assays might be explained if venom
acts on the APC in some way or if lipid autoantigens are pro-
vided by K562 cells (Fig. 2 D). In addition to using monocyte-
derived DCs, we also aimed to use an alternative system for
the generation of LC-like cells and so sorted CD34+ pro-
genitors and activated them with relevant cytokines, which
are well established to serve as a model of LC function (Hunger
et al., 2004). The cells express CD1a at moderate to high
levels (unpublished data; Porcelli et al., 1992; Sallusto and
Lanzavecchia, 1994; Yakimchuk et al., 2011) and, when added
to cultures, mediated CD1a- and venom-dependent responses
of BC2 at a ratio of 1 APC to 25 T cells (Fig. 2, E and F).
Thus, both transformed and primary CD1a+ cells were used
to formally show that T cells can be activated by bee and
wasp venom, and the primary cells validated BC2 as a re-
agent that mimics the pattern of patient responses seen ex vivo
(Fig. 1). Thus, this T cell clone could be used for the subse-
quent assays needed to isolate and identify stimulatory mole-
cules in venom.
Fractionation of venom to discover antigens
To fractionate natural venoms and discover the stimulatory
substance, we treated Apis mellifera (bee) and a mix of Vespula
Bee venom generates CD1a ligands | Bourgeois et al.
 Ar ticle

Figure 2. The autoreactive CD1a-restricted T cell clone BC2 is activated by wasp and bee venoms. Biotinylated CD1a, CD1b, and CD1c and
CD1d proteins were coated on streptavidin plates. After washing, wasp (A and B) and bee (C) venoms and dideoxymycobactin (DDM; B) were added to the
wells for 2448 h. After washing, BC2 cells (A and C) or CD8.2 cells (B) were added, and the supernatants were collected 24 h later and IFN- quantified.
(D) BC2 cells were co-cultured for 24 h with universal target cells (K562) as APCs, transfected with CD1a (K562-CD1a) or empty vector (K562) and bee
venom. IFN- in supernatants was measured by ELISA. Data are representative of three separate experiments. (E) mDCs and (F) CD34-derived LC-like cells
were incubated with IgG control or anti-CD1a antibodies for 1 h at 37C. Bee venom was then added at 2 g/ml for 1 h. BC2 cells were then added at a
1:25 APC/T cell ratio. Two representative donors out of three are shown.

species (wasp) venoms with chloroform and methanol to
create a two-phase mixture. Lipids partition into the organic
solvents (extract), whereas proteins, nucleotides and other
polar molecules are excluded (precipitate; Fig. 3 A). Nearly
all CD1-presented antigens are lipids, so it was surprising
that the lipidic extract failed to activate BC2 cells (Fig. 3 B).
Likewise, the protein-enriched precipitate failed to activate
T cells. However, T cell activation was detectable when the
extract and precipitate were recombined. This result ex-
cluded the possibility that chloroform and methanol some-
how destroyed the capacity of the whole venom to activate
BC2, and it suggested that multiple compounds within the
venom acted in concert to generate T cell response. An analo-
gous experiment performed using the K562-CD1a cellular
assay showed different results, which provided possible ex-
planations for the unexpected pattern. In contrast to the
plate assay, venom precipitate applied to intact K562-CD1a
APCs was sufficient to activate T cells. Again, extracted lipids
alone failed to activate BC2 (Fig. 3 C). These seemingly
contradictory results might be explained if venom does not
provide an activated lipid antigen, but instead provides a pro-
tein that precipitates in organic solvents and then is capable

JEM Vol. 212, No. 2 153


of producing antigenic substances when combined with
venom-derived or K562-derived lipids.
Phospholipase A2 activates T cells
In considering protein factors that might generate antigens
for CD1a proteins, it is well established that wasp and bee
venoms contain phospholipase A1 (PLA1) and A2 (PLA2).
Phospholipases are major components of venom that have
been extensively studied in regard to their antigenicity toward
MHC-restricted T cells and B cells (Aslam et al., 2006; Sin
et al., 2011). Viral phospholipases can promote activation of
NKT cells by CD1d (Zeissig et al., 2012). Based on our
preliminary experiments with recombined protein and lipid
fractions, we hypothesized that phospholipases act on venom-
derived lipids (Fig. 2) or the K562 membrane phosphodi
acylglycerols (Fig. 3) cleaving ester bonds and generating free
fatty acids, and lysophospholipids. Purified venom PLA2 from
Apis mellifera strongly activated BC2 T cells (Fig. 4 A). No T cell
activation was seen when PLA2 was exposed to T cells with
K562 cells lacking CD1a (Fig. 4 A).Therefore, the mechanism of
PLA2 on T cells is indirect and requires CD1a proteins.
Mechanism of PLA2 action
Another well-known polypeptide present in bee venom that
binds lipids and enhances PLA2 activity is mastoparan
(Argiolas and Pisano, 1983; Cabrera et al., 2011). However,
mastoparan acting alone (Fig. 4 B) did not activate BC2.
154
Figure 3. The co-incubation of both
proteins and lipids from wasp and bee
venoms triggers BC2 cells activation when
presented by CD1a protein. (A) Protein and
lipid fractions of bee and wasp venoms were
separated by organic solvent extraction using
chloroform and methanol. (B) CD1a proteins
were coated on streptavidin plates. After
washing, wasp or bee venoms and their ex-
tracts were added to the wells for 24 to 48 h.
After washing, BC2 cells were added, superna-
tants were collected after 24 h and IFN-
quantified. (C) BC2 cells were co-cultured
with universal target cells transfected with
CD1a (K562 CD1a) or empty vector (K562) and
bee venom or their extracts. Supernatants
were collected after 24 h. IFN- in superna-
tants was measured by ELISA. Data are repre-
sentative of three separate experiments.
PLA2 releases large amounts of lipid mediators of inflamma-
tion through downstream action of cyclooxygenases, which
create arachidonic acid metabolites. Therefore, testing an-
other candidate mechanism of action relating to PLA2, we
hypothesized that free arachidonic acid might bind to CD1a
proteins as an antigen or act indirectly as cyclooxygenase sub-
strate to stimulate BC2 via receptors other than the TCR.
However, we observed no arachidonic acidmediated stimu-
lation and saw dose-dependent inhibition of BC2 activation
by arachidonic acid in K562 cellular and plate assays when
added at high absolute dose (Fig. 4 C), ruling out both hy-
potheses. Although arachidonic acid could inhibit responses
at pharmacological doses, PLA2 had a reproducible and strong
stimulatory effect, suggesting that the smaller amounts of ara-
chidonic acid liberated by this enzyme do not have a domi-
nant negative effect. Finally, mDC (Fig. 4 D) and cord blood
CD34-derived LC-like cells (Fig. 4 E) act as APCs and sub-
strates of the PLA2 to activate BC2 cells in a CD1a-dependent
manner, suggesting that neo-antigens can be generated in pri-
mary APCs and that neither mDCs nor LCs express inhibi-
tory factors.
To directly test the hypothesis that bee venom PLA2 acts
by cleaving intact cellular phospholipids to create neoanti-
gens, we preincubated the enzyme with two synthetic sub-
strates for bee venom PLA2, phosphatidylcholine comprised
of singly unsaturated C18 fatty acyl chains (PC 18:1/18:1)
and phosphatidic acid with C16 fatty acyl chain in sn-1 and
Bee venom generates CD1a ligands | Bourgeois et al.
 Ar ticle

Figure 4. Bee venom PLA2 alone induces a CD1a-restricted T cell response by releasing free fatty acids. BC2 cells were co-cultured for 24 h
with universal target cells (K562) transfected with CD1a (K562 CD1a) or empty vector (K562) and bee venom or bee venom PLA2 (A), mastoparan (B), or
arachidonic acid (C). IFN- in supernatants was measured by ELISA. In (C) right panel, biotinylated CD1a proteins were coated on streptavidin plates. After
washing, bee venom or arachidonic acid were added to the wells for 24 to 48 h. After washing, BC2 cells were added for 24 h, and supernatant was sam-
pled for IFN- quantification by ELISA. Data are representative of three separate experiments. (D) mDC and (E) CD34-derived LC-like cells were incubated
with IgG control or anti-CD1a antibodies for 1 h at 37C. PLA2 was then added at 2 g/ml for 1 h. BC2 cells were then added at a 1:25 APC:T cell ratio.
Two representative donors out of three are shown.

unsaturated C18 fatty acyl chain in sn-2 (PA 16:0/18:1). We
also tested for a T cell response to products of cleavage by
PLA2 and control lipids (Fig. 5 A), including purified free
fatty acids and lysophospholipids: lysophosphatidylcholine
(LPC 18:1), oleic acid (FA 18:1), lysophosphatidic acid (LPA
18:1), and palmitic acid (FA 16:0). Higher production of
IFN- was obtained in response to phospholipids when prein-
cubated with PLA2. BC2 T cells responded to fatty acids to a
greater extent than intact phospholipids or lysophospholipids.
This result suggests that PLA2 activates CD1a-restricted T cells
by cleaving nonantigenic phospholipids into lysophospholipids
and antigenic fatty acid, in agreement with a recent study
identifying free fatty acids as CD1-presented antigens (de Jong
et al., 2014).
Venom generates lysophospholipids in vivo
Membrane phospholipids and free fatty acids are both abun-
dant in blood and tissue. However phospholipids largely
self-assemble into bilayer membrane structures, and free
fatty acids exist mainly as cytosolic metabolites or lipopro-
tein structures generated during gastrointestinal absorption.
Thus, lysophospholipids and free fatty acids are normally

JEM Vol. 212, No. 2 155


stored in macromolecular clusters and do not reach high con-
centration as free molecules in the extracellular space, where
CD1a lipid loading is thought to occur (Manolova et al.,
2003). Furthermore, although extracellular lipids, including
fatty acids, accumulate in the stratum corneum and sebaceous
glands, they are not detectable in normal dermis. Accordingly,
most models of self- or altered self-lipid display emphasize
release of free fatty acids and other antigens into the extracel
lular space in the proximity of CD1+ epidermal LC (Colonna,
2010; de Jong et al., 2010, 2014). Injection of bee and wasp
venoms into skin might locally generate such cleavage of in-
tact phosphodiacylglycerides to lysolipids and free fatty acids,
so we tested this in a human model for skin immune responses
156
Figure 5. Wasp venom phospholipase is active
in vivo in the skin. (A) Biotinylated CD1a proteins were
coated on streptavidin plates. After washing, lipids were
added at 10 g/ml for 2448 h. Lipids tested were as
follows: phosphatidylcholine 18:1 (PC18:1), lysophos-
phatidylcholine 18:1 (LPC18:1), and oleic acid 18:1 (FA
18:1; left) or phosphatidic acid 16:0 (PA16:0), lysophos-
phatidic acid 18:1 (LPA18:1), and palmitic acid 16:0
(FA16:0; right). Phosphatidylcholine and lysophospha-
tidic acid were preincubated or not with PLA2 at 50 g/ml
for 1 h at 37C. 24 h later, wells were washed and
BC2 cells were added. Supernatants were collected after
24 h. IFN- in supernatants was measured by ELISA.
Data are representative of three separate experiments.
(B) Healthy donor epidermis was injected with 10 g of
wasp venom or saline. Skin blisters were raised and, 24 h
later, the blister fluid was sampled. Lipids were extracted
with chloroform, methanol, and water using the Bligh-
Dyer method and analyzed on a quadruple time of flight
mass spectrometer. Positive mode EIC-MS detected ions
at m/z 758.5686, 786.6000, 810.5994, and 834.5993,
corresponding to the homologous series of phosphati-
dylcholine C42H81NO8P+ (16:0/18:2), C44H85NO8P+
(18:0/18:2), C46H85NO8P+ (18:2/20:2), and C48H85NO8P+
(20:4/20:2). Positive mode EIC-MS detected ions at m/z
496.3374, 524.3683, and 544.3370, corresponding to
the homologous series of lysophosphatidylcholine
C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+
(20:4). Data from one representative donor out of two
are shown.
using suction cup blisters (Salimi et al., 2013). This method
uses low pressure, sustained (60 min) suction to produce
extracellular blister fluids that are captured for immunological
and biochemical analysis before or after antigen challenge
(Salimi et al., 2013).
After injection of 10 g of venom, which mimics the
approximate volume and dose of a wasp sting, or saline vehi-
cle, 2 mm into the skin at two sites in the arm of one pa-
tient, we obtained two blister fluid samples. After extracting
the lipids from blister fluids using a mixture of chloroform,
methanol and water, we assessed for the presence of sub-
strates and products of the wasp venom PLA by mass spec-
trometry using sensitive quadrupole time of flight (QToF)
Bee venom generates CD1a ligands | Bourgeois et al.

methods (Fig. 5 B). Focusing on the main natural substrate
for PLA2, we monitored the phosphatidylcholine series for
key ions corresponding to lipids with differing fatty acid con-
tent: C42H81NO8P+ (16:0/18:2), C44H85NO8P+ (18:0/18:2),
C46H85NO8P+ (18:2/20:2), and C48H85NO8P+ (20:4/20:2;
Fig. 5 B). The masses of detected ions (m/z 758.5686,
786.6000, 810.5994, and 834.5993) matched these PC vari-
ants within the mass accuracy of the detector. Comparing
saline- and venom-injected sites, we found a twofold lower
intensity for signals corresponding with all molecular variants
of PC in the PLA2-injected skin samples, indicating that the
enzyme substantially consumed local PC (Fig. 5 B). In a sec-
ond patient, signals were reduced after venom injection for
all PC species except PC18:1 18:1 (unpublished data). Con-
versely, ions matching the lysophosphatidylcholine series,
C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+
(20:4), were readily detected in wasp venom blister fluid with
high signal but were not detected in the blister fluid from
saline injection sites. Thus, lysolipids derive from PLA itself
and not some other aspect of the suction blister model. We
measured signals corresponding to the expected masses of
free fatty acids in the positive and negative mode in these
samples, but could not detect them. Mass spectrometry de-
tection of fatty acids is less sensitive than detection of anionic
phospholipids, so we cannot derive any direct conclusions
about fatty acid concentrations in these in vivo experiments.
Nevertheless, these results demonstrate that the injection of
venom alters the local lipid content in ways that are predicted
from the specificity of PLA2 and that the cleavage reactions
needed to generate fatty acid and lysophospholipid antigens
do occur.
Direct measure of wasp and bee venom PLA bioactivity
In individual donors (Fig. 1, A and B) and cohorts (Fig. 1,
C and D) and in vitro studies with clones (Figs. 24), we noted
strikingly similar responses to bee and wasp venom, which
are currently thought to differ in their PLA subtypes, as de-
scribed above. However if fatty acids are the CD1a-lipid
ligand, similar immunological responses are expected, as fatty
acids will be generated whether PLA1 or PLA2 cleave acyl
chains from phospholipids at sn-1 or sn-2, respectively. Inter-
estingly, PLA2 has been found in venom of the neotropical
social wasps Polybia paulista (de Oliveira and Palma, 1998; dos
Santos et al., 2011) and Agelaia pallipes pallipes (Costa and
Palma, 2000). This led us to investigate whether PLA2 activ-
ity is also exerted by Vespula species of wasp venom, and fur-
thermore whether there were shared specificities between
bee and wasp venom phospholipase. Using sn-2 thiol-labeled
PLA2 substrates (arachidonyl thio-PC, heptanoyl thio-PC,
diheptanoyl thio-PC, and palmitoyl thio-PC), we observed
PLA2 activity with both bee and wasp venom indicating that
wasp venom contains PLA2 activity in addition to its known
PLA1 activity, and that specificities are partially shared with
bee venom PLA2 (Fig. 6 A). We confirmed the PLA2 activ-
ity of wasp venom could be inhibited by manoalide, a previ-
ously known PLA2 inhibitor (Fig. 6 B). Collectively, these
JEM Vol. 212, No. 2
Ar ticle
data suggest that both wasp and bee venom phospholipases
can generate common CD1a fatty acid ligands.
PLA2 induced polyclonal responses ex vivo
Having identified PLA2 as sufficient to generate CD1a-
presented antigens in vitro using T cell clones, we next sought
to determine if bee venom PLA2 is sufficient to activate poly-
clonal T cells ex vivo in cellular assays (Fig. 7). As with whole
venoms (Fig. 1), we detected higher IFN-producing cells
with the addition of 100 ng/ml bee venom PLA2 but only in
the presence of CD1a and not other CD1 isoforms (Fig. 7 A,
left). We observed significantly (P < 0.001) higher CD1a-
restricted responses in the presence of PLA2 in a cohort of 18
donors (Fig. 7 A, right). More detailed testing of an individual
responder showed that anti-CD1a antibodies prevented the
response to CD1a and PLA2 (P < 0.05), confirming the es-
sential role of CD1a (Fig. 7 B, left).
PLA2 is the essential component of venom
for CD1a ligand generation
Collectively, these studies showed that PLA2 was sufficient
to recapitulate the response to venom, so we next sought to
determine if PLA2 was necessary, or instead whether the
many known active compounds in venom could generate a
response. We observed an inhibition of IFN- production
in response to the venoms treated with neutralizing anti-
bodies that bind PLA2 (P < 0.05; Fig. 7 B, right). Further,
using manoalide we noted a blockade of response to wasp
venom and bee venom PLA2 to background levels seen with
CD1a alone (P < 0.05; Fig. 7 C) and not to lower levels,
which might be expected in the event of nonspecific toxicity
to cells. Lastly, we showed that monocyte-derived DC and
LC-like cells derived in vitro, as well as CD1a+ cells isolated
directly ex vivo from skin could induce a response by PLA2-
specific polyclonal T cells in a CD1a- and PLA-dependent
manner (Fig. 7 D). We conclude that PLA2 is a necessary
factor involved in venom-dependent activation of CD1a-
reactive T cells.
DISCUSSION
Using venoms as a model system, our data identify a new
mechanism of in vivo antigen processing by which phospho-
lipases cause local alterations in lipid content and conver-
sion of nonantigenic substances to smaller lipids, which have
CD1-mediated T cell antigenicity. Specifically, we identify
wasp and bee venom phospholipases, which act as the key
proteins that are necessary and sufficient to cleave common
cell membrane phosphodiacylglycerides, to release free fatty
acid and lysophospholipids (de Jong et al., 2014). Using the
particular clone BC2, we ruled in free fatty acids as neoanti-
gens. However, polyclonal T cells may be responding to
other lipids as well, and it is notable that CD1d presents lyso-
phospholipids to NKT cells (Gumperz et al., 2000; Fox et al.,
2009; Zeissig et al., 2012). Because the CD1a dependence of
lipid-specific T cell responses is observed in polyclonal T cells
and among many unrelated human donors, these data suggest
157

that phospholipases are important in CD1a biology, provid-
ing a potential molecular pathway underlying previous ob-
servations of T cell responses to fatty acids, which may be
important for skin barrier sensing and inflammation (de Jong
et al., 2014).
Recent studies of CD1a function have found that CD1a
autoreactive T cells and CD1a proteins are both abundantly
present within adjacent compartments of the skin (de Jong
et al., 2010, 2014). In general, there exists a physical separation
of CD1a proteins, which are mainly expressed on LC in the
epidermis, and natural autoantigens such as fatty acids, wax
esters, and squalene, which are concentrated more superfi-
cially in the sebum and cornified epithelium. The observa-
tions suggested a near neighbor model in which intact skin
prevents direct contact of autoantigens from CD1a, but skin
breach through infection or injury deposits antigenic sub-
stances more deeply within the skin so that antigens come
into contact with CD1a expressing epidermal LC (Colonna,
2010; de Jong et al., 2010; Kronenberg and Havran, 2014).
Considering bee or wasp sting as a variant of this model,
phospholipases are normally injected up to 2 mm deep into
158
Figure 6. Vespula spp and Apis mellifera venom PLA2
activity. (A) PLA2 activity was detected as free thiol groups
released from thiol-labeled lipid substrates through addition
of DNTB/EGTA and measuring color generation at 415 nm
using Apis mellifera venom with four phosphatidylcholine-
based substrates of varying acyl chain lengths and satura-
tion, but the same headgroup. Vespula spp venom PLA2
activity for substrates diheptanoyl-phosphatidylcholine and
arachidonyl-phosphatidylcholine (P < 0.001 compared with
no enzyme control). (B) Using lipid substrate with a thiol
group at the sn2 position, PLA2 activity in Vespula spp
venom in the presence or absence of manoalide was de-
tected using DNTB/EGTA, which produces a colored precipi-
tate and measured at 415 nm. Data were mean of triplicate
measurements SEM.
the skin in a process that can be meaningfully mimicked by
needle injection (Cortellini et al., 2012). Several aspects of
our data fulfill predictions that phospholipase injection might
lead to antigen generation beneath cornified epithelia in prox-
imity to CD1a proteins. Responses to venom and phospholi-
pase are detected most strongly in response to the particular
CD1 isoform (CD1a) that is most extensively expressed in the
skin. PLA substrates can derive from both venom and cellular
sources, and PLA2 would presumably have access to venom
or cell derived phospholipids during a sting. Also, although
free fatty acids could not be detected ex vivo by mass spec-
trometry, phospholipid and lysophospholipid concentrations
are demonstrated to change in suction blisters in a process that
is dependent on PLA itself. Thus, bee or wasp stings might
represent a means to locally remodel lipids and generate neo-
antigens in proximity to CD1a proteins, mimicking a natural
system of barrier sensing.
Venoms injure and thereby neutralize diverse predators
and prey through transfer of toxic substances that act on many
types of animals or insects encountered in the wild. Thus,
venoms are typically comprised of many toxic substances
Bee venom generates CD1a ligands | Bourgeois et al.

with parallel actions, and act by coopting conserved neuro-
logical or immunological mechanisms in the stung animal.
We speculate that injection of venom PLA represents a means
to coopt, through overstimulation, the natural effects of en-
dogenous mammalian PLAs in barrier sensing and immune
response. Mammalian PLA2 enzymes exist as lysosomal, cyto-
solic, and secreted (sPLA2) forms. The sPLA2 superfamily is
JEM Vol. 212, No. 2
Ar ticle
Figure 7. CD1a-restricted reactivity to
Apis mellifera venom PLA2 is found
among polyclonal T cells from blood of
healthy donors. CD3+ cells were isolated by
magnetic beads from healthy donor PBMC
and cultured for 1214 d with IL-2 and irradi-
ated K562 cells transfected with CD1a (K562
CD1a) or CD1b (K562 CD1b) or CD1c (K562
CD1c) or CD1d (K562 CD1d) or an empty vec-
tor (K562) in the presence of Apis mellifera
venom PLA2. CD1 reactivity was then exam-
ined by IFN- ELISpot with transfected or
untransfected K562 cells either in the pres-
ence or absence of Apis mellifera venom
100 ng/ml PLA2 (A, left). Data from one repre-
sentative donor of three are shown. Venom
PLA2 specific CD1a-restricted T cell responses
were measured in 18 donors (A, right). CD1a-
restricted responses were examined in the
presence or absence of anti-CD1a (B, left) for
donor C1098, and PLA2-neutralizing antibod-
ies (7B, right), and manoalide (specific PLA2
inhibitor) for wasp venom in donor C559
(C, left) and Apis mellifera venom PLA2 in donor
C334 (C, right). Representative results are
shown from three independent experiments.
(D) IFN- production was measured by
co-incubations of PLA2-specific T cells with
mDCs (left) or CD14-derived LC-like DCs
(middle) in the presence or absence of anti-
CD1a for donor C558. IFN- production was
measured from co-incubations of PLA2-specific
T cells and freshly isolated skin CD1a+ cells
in the presence or absence of anti-CD1a or
manoalide (right). Representative data from
3 (left and middle) or 2 (right) separate donors
are shown. Data are mean of triplicate mea-
surements SEM. *, P < 0.05; **, P < 0.01;
***, P < 0.001.
divided into 6 groups (I, II, III, V, X, and XII) based on
disulfide bridge structure (Starkl et al., 2013), with bee venom
PLA having the closest homology to type III human sPLA
(Scott et al., 1990; Valentin et al., 2000). sPLA2 enzymes act
in lipid digestion, host defense, and inflammation (Murakami
and Lambeau, 2013). Other evidence to suggest that mam-
malian PLAs might have CD1 or lipid-mediated effects on
159
T cells includes a recent study of mammalian PLAs from hep-
atitis virus that activate NKT cells (Fox et al., 2009; Zeissig
et al., 2012) and the role of thymic PLA in controlling NKT
cell selection (Paduraru et al., 2013). In these studies of blood-
derived T cells, CD1a autoreactivity predominates, but in
the gastrointestinal tract CD1d is highly expressed, and NKT
cells home to the intestine and liver. Thus, both studies em-
phasize a role of phospholipases, but the differing effects of
CD1a and CD1d isoforms may relate to the organ-specific
patterns of expression and T cell response. Free fatty acids
were identified as natural skin antigens for CD1a earlier this
year (de Jong et al., 2014). Collectively these studies provide
multiple lines of evidence that PLAs control various aspects
of T cell response in different organs. The clinical study of
bee and wasp sting responses represents a model system to
study the basic role of PLAs because it is characterized by
single, discreet exposures and there exist practical means to
mimic a stinging exposure and study the response in vivo and
ex vivo. Having established a role for CD1a and PLA in the
normal response to venom, future studies will likely examine
their roles in hypersensitivity and immunotherapeutic sys-
tems and take advantage of information about lysophospho-
lipids and free fatty acids as natural pathways that could be
considered for diagnosis through skin testing and antigen-
based immunotherapy.
MATERIALS AND METHODS
Isolation of human PBMC, lines, and clones. PBMCs were isolated
from healthy adult donors with a previous history of bee or wasp sting under
local ethics approval (NRES Committee South Central, Oxford C, 09/
H0606/71). T cells were purified from ficollized PBMCs using CD3 MACs
beads (Miltenyi Biotec) and cultured for 1014 d with pulsed irradiated K562-
CD1a cells in RPMI supplemented with 100 IU/ml penicillin, 100 g/ml
streptomycin, 2 mM l-glutamine, and 5% human serum (Sigma-Aldrich),
nonessential amino acids, Hepes, sodium pyruvate, and 2-mercaptoethanol
(Life Technologies) in the presence of 1 nM IL-2 (PeproTech; 5,000,000
T cells and 200,000 K562-CD1a per well of 24-well plate). CD1a reactivity was
then assessed by IFN- ELISPOT (Mabtech). ELISpot plates (Millipore)
were coated with antiIFN- antibody overnight (Mabtech). K562 cells
were pulsed with 1 g/ml wasp/bee venom (Sigma-Aldrich) or 100 ng/ml
bee venom PLA2 (Sigma-Aldrich) overnight, and were then washed and re-
suspended in R5* (RPMI supplemented with 2 mM l-glutamine, 100 IU/ml
penicillin and 100 g/ml streptomycin plus 5% human serum). The plates
were washed six times with RPMI and blocked for 1 h with RPMI supple-
mented with 2 mM l-glutamine, 100 IU/ml penicillin and 100 g/ml strep-
tomycin plus 10% human serum (R10*). 20,00050,000 T cells were added
per well to which 10,00025,000 K562 or primary cells were added. Wells
were set up in duplicate or triplicate. 10 ng/ml phorbol myristate acetate and
500 ng/ml Ionomycin was included as a positive control, and T cells alone
in the absence of K562 was included as a negative control. After overnight
incubation at 37C and 5% CO2, plates were washed 6 times in PBS-Tween
0.05% and incubated with 1 g/ml of biotin-linked antiIFN- monoclonal
antibody (Mabtech) for 2 h. After washing 6 times in PBS-Tween 0.05%,
the plates were incubated for an additional 1 h with streptavidin-alkaline
phosphatase (Mabtech). Spots were visualized using an alkaline phosphatase
conjugate substrate kit (Bio-Rad Laboratories) and enumerated using an au-
tomated ELISpot reader (Autimmun Diagnostika GmbH). The T cell lines,
BC2 and CD8-2, as well as K562-CD1 cells have been described previously
(Partners Health Care Institutional Review Board; Rosat et al., 1999; de Jong
et al., 2010, 2014).
160
Isolation and generation of human APCs. PBMCs were incubated
with GM-CSF (300 U/ml) for 30 min. After removing the nonadherent
cells, medium with GM-CSF (300 U/ml) and IL-4 was added (200 U/ml)
for 34 d. Adherent mDCs were collected by addition of PBS/0.5 mM of
EDTA for 20 min, and then irradiated by -irradiation (5,000 rad). High
CD1a expression was verified by flow cytometry. We generated LC-like
cells in vitro using two published protocols. Starting with CD14+ monocytes
from human buffy coats from the NHS Blood Bank ( John Radcliffe Hospi-
tal, Oxford, UK), CD14+ human cells were isolated using MACS cell sepa-
ration (Miltenyi Biotec) according to the suppliers instructions. In brief,
such in vitro LC-like cells were prepared as previously described (Caux et al.,
1997; Geissmann et al., 1998) from CD14+ cells, which were cultured in
6-well plates in complete medium in the presence of IL-4 (250 ng/ml;
PeproTech), GM-CSF (100 ng/ml; PeproTech), and TGF-1 (10 ng/ml;
PeproTech). At days 2 and 4, cultures were replated in the presence of
the aforementioned cytokines to generate cells which were 1015% CD1a+
CD207+. The in vitro LC-like cells were incubated with 10 g/ml anti
HLA-ABC and antiHLA-DR blocking antibodies (W6/32 and L243,
respectively) for one hour before co-culture with T cells, to minimize HLA-
restricted responses. Alternatively, samples of human cord blood were ob-
tained from CFAR Virology Core Laboratory at UCLA (Los Angeles, CA).
CD34+ human progenitor cells were isolated using MACS cell separation
(Miltenyi Biotec) according to the suppliers instructions. LCs were then
prepared as previously described (Hunger et al., 2004) but with some modi-
fications. Specifically, cultures of CD34+ cells were established in the pres-
ence of stem cell factor (25 ng/ml; R&D Systems), GM-CSF (200 U/ml;
Sanofi US Services, Inc.), and TNF (2.5 ng/ml; Thermo Fisher Scientific).
At day 5, cultures were replated in the presence of GM-CSF (200 U/ml;
Sanofi US Services, Inc.) and TGF-1 (1 ng/ml; R&D Systems) to in-
crease CD1a expression. LC were harvested at day 12. The LC quality of
the cells was checked by flow cytometry, and found to consist of 3040%
CD1a+CD207+ cells.
Isolation of CD1a+ cells from skin. After removing subcutaneous fat,
skin sections were cut into 5-mm-wide pieces and cultured in 2 mg/ml
dispase solution at 4C overnight. The epidermis was then separated from
the dermis and cultured in complete media for 5 d. Migratory adherent cells
were harvested and enriched by density gradient centrifugation, and CD1a+
cells were isolated using MACs cell separation (Miltenyi Biotec). The CD1a+
cells were incubated with 10 g/ml antiHLA-ABC and antiHLA-DR
blocking antibodies (W6/32 and L243, respectively) for 1 h before co-culture
with T cells, to minimize HLA-restricted responses.
CD1-plate assays. Biotinylated CD1a, CD1b, CD1c, and CD1d proteins
(National Institutes of Health Tetramer Core Facility, Emory University,
Atlanta, GA) and anti-CD11a antibodies were added on streptavidin-coated
plates (Thermo Fisher Scientific) at 10 g/ml and 2.5 g/ml, respectively, for
24 h at room temperature as detailed previously (de Jong et al., 2014). Wasp
and bee venoms, their lipidic extracts, dideoxymycobactin (DDM), arachi-
donic acid, phosphatidylcholine 18:1, phosphatic acid 16:0, lysophosphatidyl-
choline 18:1, lysophosphatidic acid 16:0 or fatty acids 18:1, and 16:0 (Avanti)
were loaded at 10 g/ml in PBS for 2448 h at room temperature. BC2 cells
and CD8-2 cells were then added to the well at 100,000 cells/well.
Cellular assays. 150,000 BC2 cells were co-cultured with 20,000 K562-
CD1 cells for 24 h with wasp and bee venoms, chloroform and methanol
extracts, chloroform and methanol precipitates, PLA2 (Sigma-Aldrich), mas
toparan (Sigma-Aldrich) or arachidonic acid (Sigma-Aldrich). In some experi-
ments 10 g/ml anti-CD1a blocking antibody (OKT-6), or 10 g/ml IgG1
isotype control (P3), or 100 M manoalide, an inhibitor of PLA2 that acts
by covalently modifying lysine residues (Enzo Life Sciences), were added to
K562 or primary cells before addition of T cells.
Venom extraction. 120 g of lyophilized wasp (Vespula species; ALK) and
bee venom were resuspended in a solution of (1:1) methanol/chloroform.
Bee venom generates CD1a ligands | Bourgeois et al.

After 40 min of rotation and a centrifugation at 300 g for 5 min, supernatant
was collected. Lipid enriched-supernatant and protein-enriched precipitates
were collected and resuspended in 1.2-ml of sterile PBS and normalized to
the input mass, or when possible, weighed to determine the exact mass.
Blister fluids. Healthy donors were injected with 10 g of wasp venom or
saline intraepidermally, a dose which is at the low end of venom normally ad-
ministered in a sting (ALK). Suction was applied to the skin at 200 mmHg for
1 h, which induced a split between the epidermis and dermis (Salimi et al.,
2013). The blister fluids were collected by aspiration at 24 h and immediately
stored at 20C. Lipids were extracted with chloroform, methanol, and
water using the Bligh-Dyer method (Bligh and Dyer, 1959).
HPLC-(electrospray ionization)-quadrupole time of flight mass
spectrometry. The analyses were performed using an Agilent Technologies
6520 ESI-QTof coupled with HPLC (Agilent Technologies 1200). Lipids
were separated using a 3 m 150 mm 2 mm diol column (Varian) as pre-
viously described (Layre et al., 2011). Spectra were collected in positive mode
from 100 to 3,000 m/z at 1 spectrum/s. Internal calibrations were performed
using calibrants at 121.050873 m/z and 922.009798 m/z that were coinfused
into the spray chamber and used to monitor the stability of ion signal. Data
were analyzed using MassHunter Workstation Qualitative Analysis Software
(Agilent Technologies).
ELISA. Culture supernatants were harvested and tested for IFN- produc-
tion by ELISA using the clone 2G1 (Thermo Fisher Scientific) for coating at
2 g/ml and the biotin-labeled clone B133.5 (Thermo Fisher Scientific) at
0.1 g/ml. Streptavidin-HRP (BD) was incubated for 30 min and the sub-
strate was revealed by 2,2-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-
diammonium salt (ABTS).
PLA2 biochemical activity experiments. PLA2 activity in wasp and bee
venom were detected using site-specific substrate kit (Cayman Chemicals),
according to manufacturers instructions. In a flat-bottom 96-well plate, 50 ng
wasp or bee venom or 10 ng PLA2 (Sigma-Aldrich), plus 5 l assay buffer,
were incubated for 1 h at RT with a substrate solution. Substrates used have
a thiol group attached at the sn-2 position of the phospholipid, and included
arachidonyl thio-PC, heptanoyl thio-PC, diheptanoyl thio-PC, and palmi-
toyl thio-PC. In the presence of PLA2, cleavage of the substrate at the sn-2
position resulted in release of the thiol group. After 1 h incubation, 10 l
DNTB/EGTA was added, which reacts with free thiol groups producing a
colored precipitate, which was measured at 415 nm wavelength (iMark
Microplate Reader; Bio-Rad Laboratories).
Statistics. Cohort of healthy donors investigated for CD1a-restricted wasp
venom, bee venom, and PLA2-specific responses were analyzed using one-
tailed Wilcoxon matched-pairs signed rank test. All other polyclonal T cells
responses were analyzed using one-tailed Mann-Whitney tests.
We thank John Altman and the NIH tetramer facility for providing recombinant
CD1 proteins.
This work was funded by the MRC and NIHR Biomedical Research Centre, the
National Institutes of Health (NIH; NIAMS R01 048632), and the Burroughs
Wellcome Foundation Program in Translational Medicine. GO also acknowledges the
support of the National Institute for Health Research Clinical Research Network.
The work is supported by Cancer Research UK (Programme Grant C399/A2291
to V. Cerundolo).
The authors have no conflicting financial interests.
Submitted: 7 August 2014
Accepted: 11 December 2014
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INSIGHTS

T cells require DOCK8 for flexibility and function

In this issue, Zhang et al. report a novel function for DOCK8 (dedicator of cytokinesis 8) in controlling the
structural integrity of lymphocytes. DOCK8 deficiency severely compromised survival of T cells during
trafficking through dense tissue networks, impairing skin-specific protective antiviral immune responses.
Identifying the genetic lesions underlying primary immunodeficiencies has an immediate impact on
disease diagnosis, therapy, and patient management. It can also provide mechanistic explanations for disease
pathogenesis, such as the absence of T and NK cells in X-SCID due to the requirement for gc in IL-7R and
IL-15R signaling, and the lack of switched Ig isotypes in hyperimmunoglobulin M syndrome due to the
Insight from requirement for CD40/CD40L interactions in this process. However, there are other examples where iden-
Stuart Tangye tifying the mutant gene in a particular disease opens up a new field of study, for the simple reasons that little
was known about the function of this gene in immune cells. The discovery that biallelic loss-of-function
mutations in DOCK8 causes autosomal recessive hyper IgE syndrome falls into this second category. DOCK8 is a guanine
nucleotide exchange factor that activates small GTPases such as Cdc42. Previous studies revealed that cytoskeletal defects in
DOCK8-deficient B cells, T cells, and NK cells impairs their effector function. However, it is not clear why individuals
with DOCK8 mutations specifically develop severe viral infections of the skin. Zhang et al. theorized that since DOCK8-
deficient lymphocytes exhibited normal chemotaxis and initial migration into human tissue, these cells may be impaired
in their ability to traffic through the dense tissue networks of the
skin. By analyzing humans, mice, and cell lines, DOCK8-
deficient T and NK cells were found to have aberrant mor-
phologies under conditions that replicated skin infiltration
and penetration, and this ultimately contributed to an unusual
form of cell death termed cytothripsis (cell shattering). This
morphological effect was replicated by abolishing expression
of Cdc42 or p21-activated kinase (PAK), but not RAC1/2 or
the WAS protein, thereby establishing that DOCK8 operates
in this setting by activating these regulators of actin polymer-
ization. Overall, the stress experienced by DOCK8-deficient In adoptive transfer experiments, Dock8-deficient T cells were
cells moving through dense tissue networks abrogated the gen- undetectable in skin one month after HSV infection compared with
eration and maintenance of tissue-resident memory CD8+ normal numbers of control T cells. Left panel: two photon imaging
T cells that are important for protective immunity at such sites. of the skin 17 days after infection with HSV; right panel: numbers of
This selective inability of effector T cells lacking HSV-specific T cells in skin at various times after infection with HSV.
DOCK8 to efficiently localize to tissues high in collagen
content and provide potent antiviral immunity at these sites may explain why DOCK8-deficient patients have heightened
susceptibility to skin-trophic viral infections, yet systemic viral infections are more effectively controlled. These findings reveal the
morphological flexibility of immune cells that is required for them to execute effector function in nonlymphoid tissues and the
critical function of DOCK8/Cdc42/PAK in this process. It will be important to elucidate the mechanism by which DOCK8
integrates into TCR signaling, as well as to assess virus-specific skin-resident memory cells in DOCK8-deficient humans to
establish a paucity of these cells at these sites. Identification of the components that regulate lymphocyte integrity, motility,
and survival under conditions of migratory stress may provide an opportunity to enhance tissue-specific immunity and memory
in patients with germline mutations in this pathway. Eventually, shedding light on the pathology arising in the skin may result
in improved outcomes for patients with this often-fatal immunodeficiency.
Zhang, Q., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20141307.

Stuart G. Tangye, Garvan Institute of Medical Research: s.tangye@garvan.org.au

 Article

DOCK8 regulates lymphocyte shape

integrity for skin antiviral immunity
Qian Zhang,1 Christopher G. Dove,1* Jyh Liang Hor,5,6*
Heardley M. Murdock,1 Dara M. Strauss-Albee,1 Jordan A. Garcia,1
Judith N. Mandl,2 Rachael A. Grodick,1 Huie Jing,1
Devon B. Chandler-Brown,1 Timothy E. Lenardo,1 Greg Crawford,7
Helen F. Matthews,3 Alexandra F. Freeman,4 Richard J. Cornall,7
Ronald N. Germain,2 Scott N. Mueller,5,6 and Helen C. Su1
1Laboratory of Host Defenses, 2Laboratory of Systems Biology, 3Laboratory of Immunology, and 4Laboratory of Clinical
Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
5Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, and 6The ARC Centre

of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria 3010, Australia
7MRC Human Immunology Unit, Nuffield Department of Medicine, Oxford University, Oxford OX3 7BN, England, UK

DOCK8 mutations result in an inherited combined immunodeficiency characterized by in-

creased susceptibility to skin and other infections. We show that when DOCK8-deficient
T and NK cells migrate through confined spaces, they develop cell shape and nuclear defor-
mation abnormalities that do not impair chemotaxis but contribute to a distinct form of
catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration
in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is
unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells
prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs
control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape
integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

CORRESPONDENCE
Helen C. Su:
hsu@niaid.nih.gov
OR
Scott N. Mueller:
smue@unimelb.edu.au
Abbreviations used: DNFB,
2,4-dinitrofluorobenzene; GEF,
guanine nucleotide exchange
factor; PAK, p21-activated
kinase; PI, propidium iodide;
TRM, tissue-resident memory
CD8 T cells; WASP, Wiskott-
Aldrich syndrome protein.
DOCK8, which is highly expressed only within
the immune system, functions as an atypical gua-
nine nucleotide exchange factor (GEF) to acti-
vate small Rho GTPases (Ct and Vuori, 2002;
Ruusala and Aspenstrm, 2004; Meller et al.,
2005; Harada et al., 2012; Mou et al., 2012) and
its role as an adaptor in TLR9-MYD88 signal-
ing suggests additional functions beyond GEF
activity ( Jabara et al., 2012). DOCK proteins and
their orthologs participate in diverse biological
processes, including gonadal and epidermal cell
migration during embryonic development, tumor
cell invasion, and leukocyte chemotaxis and traf-
ficking through LNs (Kunisaki et al., 2006; Ct
and Vuori, 2007; Gotoh et al., 2008; Kikuchi
et al., 2008; Nishikimi et al., 2009, 2013; Harada
et al., 2012).
For most people without any obvious im-
mune deficiency, infections with HSV, varicella-
zoster virus, or human papillomavirus cause
self-limited cold sores, chickenpox, or warts. How
ever, these viruses can reemerge from latency to
*C.G. Dove and J.L. Hor contributed equally to this paper.
cause disease in up to 30% of the population
(Higgins et al., 1993; Kilkenny and Marks, 1996;
Harpaz et al., 2008). In contrast to normal individ
uals, DOCK8-deficient patients with autosomal-
recessive loss-of-function mutations in DOCK8
have impaired cellular and humoral immunity
(Engelhardt et al., 2009; Zhang et al., 2009; Su
et al., 2011; Jing et al., 2014) that manifests as ex-
treme susceptibility to skin and other infections
(Chu et al., 2012). Patients often suffer from
disseminated and persistent viral skin infections
including those caused by HSV, varicella-zoster
virus, human papillomavirus, and molluscum
contagiosum. Their chronic viral infections may
reflect multiple defects that affect T cell activa-
tion, proliferation, survival, and priming by den-
dritic cells (Zhang et al., 2009; Lambe et al., 2011;
Randall et al., 2011; Harada et al., 2012; Crawford
et al., 2013), NK cell cytotoxicity (Ham et al.,
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J. Exp. Med. 2014 Vol. 211 No. 13 25492566 2549
www.jem.org/cgi/doi/10.1084/jem.20141307

2013; Mizesko et al., 2013), and antiviral cytokine production
(Zhang et al., 2009).
T effector cells are a critical component of immunity to the
types of viral skin infections characteristically seen in DOCK8
deficiency. These cells must scan for and target pathogens
within the large volume of the skin, which is organized into
two layers. The epidermis is composed of interlocking arrays
of keratinocytes that impede the passage of immune effector
cells (Honda et al., 2014). In contrast, the dermis is composed
of a dense network of packed collagen fibers, through which
immune cells must navigate (Wolf et al., 2009; Honda et al.,
2014). The collagen fibers make up as much as one third of
the wet weight of skin, as compared with 10% of aorta or
1% or less of other organs such as spleen and brain (Lowry
et al., 1941; Neuman and Logan, 1950).Thus, the extracellular
environments of the epidermis and dermis are characterized
by many highly confined spaces, which are likely to tax the
structural integrity of cells navigating to their targets. Given
the presumptive role of DOCK8 in controlling cell cytoskel-
etal function and migration capacity, the fact that DOCK8-
deficient patientsin comparison with other combined
immunodeficiency patientsseem to suffer disproportion-
ately from a broad variety of skin infections, and the evidence
for physical constraints on immune cell movement in skin, we
investigated whether the skin viral susceptibility of these pa-
tients might relate to a defect in effector cell migration. Our
studies revealed an unexpected, critical role for DOCK8 in
maintaining lymphocyte cellular integrity during migration
in dense environments that limits host resistance.
RESULTS
DOCK8-deficient T cells and NK cells develop abnormally
elongated shape and nuclear deformation
Despite their susceptibility to skin infections including HSV
(Fig. 1 A), DOCK8-deficient patients have histologically
2550
Figure 1. Skin disease in DOCK8-defi-
cient patients. (A) Chronic ulcerating and
disseminated HSV infections of the skin in-
volving axilla (left), trunk (middle), and vulva
(right) of a DOCK8-deficient patient. 19 of
34 patients in the NIH cohort had similar re-
current or disseminated HSV skin infections.
(B) Skin histology in human DOCK8 deficiency.
Hematoxylin and eosin staining showing epi-
dermal and dermal layers of skin biopsies from
normal healthy control (left) and DOCK8-
deficient patient (right). Note highly packed,
multilayered epithelial cells in the epidermis
and many large pink collagen bundles in the
dermis. Bars, 1 mm. Skin biopsies were evaluated
from 17 DOCK8-deficient patients. (C) T cells
(green) from either a healthy control (left) or
DOCK8-deficient patient (right), shown mi-
grating in dermis (purple) of foreskin tissues
from healthy donors. Red arrows, elongated
cell processes. Bars, 10 m. Shown is a repre-
sentative of four patients and four healthy
controls tested in three experiments.
normal skin structures (Fig. 1 B), likely reflecting the fact that
DOCK8 is not expressed by normal keratinocytes, fibroblasts,
and endothelial cells (Su et al., 2011). Dock8-deficient den-
dritic cells migrate poorly into LNs (Harada et al., 2012). This
raised the possibility that impaired presentation of viral antigens
by dendritic cells within draining LNs might lead to defective
T cell immunity to viruses that infect the skin. However, given
that DOCK8 is expressed in T cells as well as myeloid cells,
it was also possible that a similar migration defect among skin-
infiltrating cytotoxic lymphocytes might impair their effec-
tive ability to control viral replication locally. To investigate
the motility of DOCK8-deficient T cells, we examined fluor
escently labeled T cells from patients that were allowed to
migrate into the dermal layer of human foreskin biopsies. The
mutant T cells moved through the extracellular matrix of the
skin but had abnormally elongated thin processes (Fig. 1 C).
A similar phenotype was observed when the skins dermal layer
was modeled in vitro by placing patient T cells into a three-
dimensional (3D) collagen gel matrix (Fig. 2 A; and Video 1);
this differed from the rounded, nonmotile phenotype reported
for Dock8-deficient dendritic cells (Harada et al., 2012).
The elongated T cells also had dramatically elongated nuclei
(Fig. 2 A). Such elongation was not due to failed cytokinesis
despite a role of some DOCK proteins in promoting cell di-
vision (Kittler et al., 2007) because elongated cells only had
one centrosome (Fig. 2 B), elongation persisted at high levels
when cell cycle progression was minimal after prolonged cul-
ture (Fig. 2 C), and cell cycle arrest at G1/S did not prevent
elongation (Fig. 2, D and E). Time-lapse video microscopy re-
vealed that T cells from patients (Fig. 2 F) spent an increased
proportion of time elongated, as did normal T cells in which
DOCK8 expression was silenced by transfection with siRNA
(Fig. 2 G). Abnormal elongation also occurred in T cells and
NK cells from Dock8 mutant mice (Fig. 2, H and I; and Video 2).
Like T cells, NK cells are present in the skin of healthy
DOCK8 and cytothripsis in skin viral infections | Zhang et al.

persons and those with inflammatory skin conditions (Ebert
et al., 2006; Grgoire et al., 2007). These lymphocytes also help
protect against HSV and other viral infections, especially at
early times after infection (Biron et al., 1999). Thus, the in-
ability to maintain shape integrity during migration in the 3D
microenvironment was an intrinsic property of the lympho-
cytes due to the lack of DOCK8 expression.
DOCK8-deficient cells exhibit normal chemotaxis
but undergo fragmentation and cell death in 3D conditions
The random migratory pattern of T cells within collagen gel
matrices mimics their migration pattern in skin tissues, facili-
tating surveillance for infected cells. We saw that DOCK8-
deficient T cells migrating within 3D collagen gel matrices
JEM Vol. 211, No. 13
Ar ticle
Figure 2. Abnormal morphology of
DOCK8-deficient lymphocytes when
migrating in 3D collagen gel matrices.
(A) Confocal imaging of T cells migrating in a
collagen gel matrix (3.6 mg/ml) with nuclear
staining. BF, brightfield. DAPI, blue. Represen-
tative of six experiments. (B) Confocal imaging
after in-gel immunofluorescence staining of
control and patient T cells for pericentrin (red,
indicated by arrows), -tubulin (green), and
nuclei (blue). Stained gels were physically
compressed before imaging. Left panels: mi-
grating cells, each showing one centrosome.
Right panels: dividing anaphase cells, each
showing two centrosomes. Representative of
three patients and three healthy controls from
three experiments. (C) Proportions of resting
T cells that were abnormally elongated. Less
than 2% of cells were in S/G2/M phases after
6 wk of culture. Three controls and three pa-
tients were tested from three experiments.
(D) Proportions of patient T cells that were
abnormally elongated after cell cycle blockade.
Similar analysis as for C, except that cells were
from three patients that were treated with
either aphidicolin (Aph) or vehicle (DMSO),
tested in three experiments. (E) Nuclear elon-
gation among abnormally elongated cells.
Nuclei in cells from D were stained with
Hoechst for analysis. (F) Percentage of time
cells spent elongated in the collagen matrices.
6 patients and 12 healthy controls were tested
from five experiments. (G) Similar analysis as
for F, except performed with T cells transfected
with DOCK8 or nonspecific (NS) siRNA, from
four experiments. Representative immunoblot
showing DOCK8 knockdown efficiency.
(H and I) Proportions elongated of T cells (H)
and NK cells (I) from 10 Dock8-deficient (KO)
or control (WT) mice from three experiments,
migrating in collagen gel matrices. Bars,
10 m. CI show means SD. Unpaired two-
tailed Students t test were performed. Statis-
tical significance indicated by *, P < 0.05;
****, P < 0.0001; ns, not significant.
occasionally remained in place while moving both ends in a
poorly coordinated manner, suggesting that immune surveil-
lance could potentially be compromised (Video 1). However,
during skin infection, various chemokines are induced (Stock
et al., 2014), which could contribute to directed T cell migra-
tion toward viral pathogens.To test whether the abnormal mor-
phology of Dock8-deficient T cells was associated with impaired
chemotaxis, we tracked individual cells moving through col-
lagen matrices toward a gradient of CXCL12 (SDF-1). For
each cell, directional velocity and track straightness toward the
chemokine, as well as speed along the path traveled and track
straightness from origin to destination, were analyzed over a
30-min period (Fig. 3 E). The mean values for the population
of cells from each DOCK8-deficient patient were similar to
2551

normal healthy control cells (Fig. 3, AD). Chemotaxis was also
unaffected when tested using normal T cells in which DOCK8
expression was silenced after transfection with siRNA (Fig. 3,
AD, and F). Thus, DOCK8-deficient cells were capable of
normally sensing and migrating toward a chemokine source.
In contrast, after hours of moving within the matrix, pa-
tient T cells often fragmented catastrophically as they moved
in place (Fig. 4 A and Video 3). The cell fragments contained
pieces of deformed nucleus, as shown by propidium iodide
(PI) staining. Flow cytometric quantification of dead and dying
cells revealed that silencing DOCK8 in otherwise healthy donor
human T cells caused them to die within the gels but not within
liquid medium (Fig. 4 B). T cells and NK cells, from patients
or mice genetically deficient in DOCK8, showed a similar fate
(Fig. 4 C and not depicted). Cells that died had spent slightly
greater proportion of time elongated, but the duration of
elongation episodes immediately preceding fragmentation
were longer, suggesting that abnormal morphology correlated
with cell death (Fig. 4, D and E). The dying cells recovered
from collagen gels showed ultrastructural features reminiscent
of apoptosis and necrosis with cell shrinkage, loss of microvilli
but no membrane blebbing, and holes in the plasma mem-
brane (Fig. 5 A) but lacked biochemical evidence of classical
2552
Figure 3. Chemotaxis of DOCK8-deficient T cells
migrating toward CXCL12 in collagen gel matrices.
(AD) Velocity toward chemokine (A), chemokine direc-
tionality (B), track speed (C), and directionality (D). Each
symbol represents the means of these values for indi-
vidually tracked cells from each sample. The horizontal
line designates the average of these means. T cells from
four patients and eight controls were evaluated from
four experiments. In two separate experiments, normal
T cells were transfected with DOCK8 siRNA or nonspecific
(NS) siRNA. (E) Cartoon showing measurements of che-
motaxis, based upon the actual route that a cell took as
indicated by the gray dotted line. (F) Representative flow
cytometric measurement of CXCR4 (receptor for CXCL12)
expression on cells used in the chemotaxis assays. Un-
paired two-tailed Students t tests were performed. All
statistical comparisons were nonsignificant.
apoptosis such as loss of mitochondrial membrane potential
or caspase activation (Fig. 5, BE). This cell death was not
blocked by treatment with the pan-caspase inhibitor zVAD-fmk
(Fig. 5 F). Together, these results establish that DOCK8-
deficient lymphocytes, when moving for prolonged periods in
a 3D environment, undergo a distinct form of cell death asso-
ciated with abnormal cell shape and movement, which we
term cytothripsis (cell shattering).
Abnormal elongation requires movement
through confined spaces but not adhesive forces
We next investigated the spatial configuration of the micro-
environment that elicited this abnormal phenotype. Lowering
the concentration of collagen within the gel matrices, which
increases the average pore size through which the cells mi-
grate (Pedersen and Swartz, 2005), resulted in a correspond-
ing decrease in the amount of cell death (Fig. 6 A). Abnormal
elongation with nuclear deformation also occurred when pa-
tient T cells migrated through the small pores of an uncoated
polycarbonate transwell insert (Fig. 6 B) or within 3D matri-
ces composed of agarose (Fig. 6 C). This phenotype was not
observed when cells moved on ICAM-coated (Fig. 6 D) or
DOCK8 and cytothripsis in skin viral infections | Zhang et al.
 Ar ticle

Figure 4. Elongation when migrating through confined spaces leads to cytothripsis. (A) Time-lapse microscopy of a T cell from a DOCK8-deficient
patient migrating in collagen matrix. PI (orange) added when indicated. Shown is a representative of three patients and three healthy controls, from three
experiments. (B) Flow cytometric quantification of dead and dying cells by Annexin V and PI staining, after collagenase treatment to recover unstained
cells. Normal human T cells were transfected with either DOCK8 (open symbols) or nonspecific (NS) siRNA (filled symbols), and allowed to migrate in col-
lagen matrices (solid line) or medium (dotted line) for the indicated times. Shown is a representative of three experiments. (C) Similar analysis as for B,
after migration in medium or collagen for 24 h, of NK cells from six Dock8-deficient (KO) or control (WT) mice from three experiments. (D) Percentages of
time spent elongated, for those Dock8-deficient T cells that died or remained alive after migrating in collagen matrix for 16 h. (E) Similar to D, except that
duration of elongation episodes was measured for each single episode of elongation that immediately preceded the cell fragmenting (F), or for all other
elongation episodes (NF). Bar, 20 m. Three patients were tested in D and E from three experiments. CE show means SD. Two-way ANOVA was per-
formed for C. Unpaired Students t test was performed for D and E. Statistical significance indicated by *, P < 0.05; ***, P < 0.001; ns, nonsignificant.

collagen-coated 2D (Fig. 6 E) flat surfaces. Although integrin-
mediated strong adhesive forces are not normally required for
leukocyte locomotion within a 3D environment (Lmmermann
et al., 2008), the sometimes tethered appearance of elongated
DOCK8-deficient cells (Video 1) raised the question of whether
such adhesion contributed to the abnormal phenotype. How-
ever, this explanation seemed unlikely because agarose and
polycarbonate are not ligands for integrin receptors yet could
elicit the abnormal phenotype (Fig. 6, B and C). Elongation was
also unaffected by disrupting interactions between integrin
1 collagen receptors on patient cells with collagen-containing
gel matrix by either adding anti-integrin 1 subunit antibod-
ies to the collagen gel matrix (Fig. 6 E) or silencing its expres-
sion within T cells from a DOCK8-deficient patient (Fig. 6 F).
Together, these results argue that the abnormal shape integ-
rity of DOCK8-deficient cells was elicited solely by the con-
fined spaces that constrained cell movement. This could explain
why the cellular phenotype could vary from tissue to tissue in
the body, depending on their physical characteristics, and why
disease in the patients manifests as an immunodeficiency with
disproportionate involvement of skin, whose tight adhesive
junctions between epidermal cells and tight spacing between
dermal collagen bands demand marked lymphocyte shape de-
formation for effective migration.
DOCK8, through CDC42 and p21-activated kinase (PAK),
regulates lymphocyte shape integrity to coordinate
cytoskeletal structures during cell movement
Lymphocyte migration in the skin presents a challenge espe-
cially for the nucleus, which is normally the largest and least
deformable organelle (Friedl et al., 2011). Cell body shape
change must at some level be coordinated with nuclear shape
change during cell migration. Given the elongation phenotype
of DOCK8-deficient cells described above, DOCK8 likely
regulates this architectural machinery. The small Rho GTPases
CDC42 and RAC serve as molecular switches to control di-
verse biological processes, including cell morphogenesis and
migration ( Jaffe and Hall, 2005); moreover, DOCK8 can ac-
tivate CDC42 and RAC (Harada et al., 2012; Mou et al.,
2012), regulating CDC42 to facilitate dendritic cell migration
(Harada et al., 2012). We therefore investigated the possible
contribution of these small GTPases to the DOCK8-deficient
phenotype of lymphocytes. Knockdown of CDC42 but not
RAC1/2 in T cells from normal donors recapitulated the
DOCK8-deficient phenotype of cell elongation, nuclear de-
formation, and cell death (Fig. 7, AC). CDC42 in turn acti-
vates multiple effectors, including PAK and Wiskott-Aldrich
syndrome protein (WASP). Treatment of normal T cells with
the class I PAK small molecule kinase inhibitor IPA3, or with

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Figure 5. Cytothripsis is biochemically distinct from apoptosis.
(A) Scanning electron micrographs of control or patient T cells after mi-
grating in collagen gel matrices. (B) Mitochondrial membrane potentials
of patient or control T cells migrating in collagen gels. A decrease in the
ratio of red (JC-1 aggregates) to green (JC-1 monomer) is indicative of
loss of potential. (C) Proportions of T cells expressing active caspase-3
after migrating in collagen gels for the indicated times. Eight controls and
four patients were tested from four experiments. (D and E) Immuno
blotting for highly active cleaved caspase-8 (D) and caspase-9 (E) proteins.
Lysates were prepared from control or patient T cells that had migrated in
medium (0) or collagen gels for the indicated times. The black line shows
that intervening lanes were spliced out. (F) Inability to block cytothripsis
with the pan-caspase inhibitor zVAD. Annexin V+ or PI+ staining of mi-
grating cells (nine controls, four patients, from four experiments) was
quantitated by flow cytometry after recovery of unstained cells from gels
2554
PAK1/2 siRNA, also phenocopied loss of DOCK8 or CDC42
(Fig. 7, DF), whereas loss of WASP expression in T cells
showed no such effect (Fig. 7, G and H). Similar results were
seen for T cells from a Wiskott-Aldrich syndrome patient or
Was KO mice (unpublished data). Loss of DOCK8, CDC42,
or PAK1/2 expression or function did not reciprocally decrease
each others protein levels (unpublished data; Akbar et al., 2011).
These results suggest that DOCK8 acts proximally with CDC42
and PAK in regulating lymphocyte shape integrity, most likely
through complex spatial and temporal effects on actin, myo-
sin, and microtubule cytoskeletal structures (Bokoch, 2003;
Li and Gundersen, 2008). Indeed, the absence of DOCK8,
CDC42, or PAK activity resulted in cytoskeletal discoordina-
tion between the front and rear of migrating cells, seen as ab-
normal cellular elongation with slightly decreased total F-actin
polymerization present at both poles and abnormal position-
ing of the microtubules including the microtubule organizing
center (Fig. 8).
Defective cell integrity occurs
during response to viral skin infections
Viral replication in the skin occurs primarily within keratino-
cytes in the epidermis; however, some viruses also infect der-
mal cells such as fibroblasts or neurons at the epidermal-dermal
junction (Cunningham et al., 1985; Drijkoningen et al., 1988;
Muraki et al., 1992; Nikkels et al., 1996). This places special
demands on effector immune cells that must successfully nav-
igate into and through dermis after exiting dermal vessels and
then migrate further into the densely connected cell layers of
the epidermis to mediate host defense. To assess the function
of Dock8-deficient T cells during a viral infection, we infected
mice with HSV, which is the only virus causing severe skin in-
fection in patients that is also capable of infecting mice. When
HSV is inoculated in the epidermis on the flank, it establishes
infection within the dorsal root ganglion before spreading
through the nerves, from which it emerges to cause a skin rash
in a dermatomal distribution (Fig. 9 A; Simmons and Nash,
1984). Containment of the rash reflects the ability of CD8
T cells to limit secondary spread of the virus down the flank
(van Lint et al., 2004). Upon acute infection, Dock8-deficient
mice developed herpetic skin disease that was more severe with
increased mortality when compared with WT mice (Fig. 9,
AC). To visualize T cells that were responding within the in-
fected skin, we adoptively transferred Dock8-deficient or con-
trol WT T cells into normal mice. The recipient mice were
with collagenase. All panels show a positive control in which apoptosis
was induced in healthy donor T cells within the collagen gels using anti-
FAS antibodies with Protein A cross-linking. Cells were analyzed after
migrating within the collagen gels for the indicated times or as described
in the Materials and methods. A, B, D, and E show representatives of three
tested patients and controls from three experiments. Bars, 10 m. C and F
show means SD. Unpaired Students t test was performed for C and F
(right), and two-way ANOVA was performed for F (left). Statistical signifi-
cance indicated by *, P < 0.05; ****, P < 0.0001; ns, nonsignificant.
DOCK8 and cytothripsis in skin viral infections | Zhang et al.
 Ar ticle

Figure 6. Requirement for migration through confined spaces but not for adhesion in eliciting loss of shape integrity. (A) Proportions of
T cells (nine controls, four patients, from four experiments) that were Annexin V+ or PI+, after migration in increasing collagen concentrations for 24 h in
collagen gels of increasing matrix density. (B) Confocal and diffusion interference contrast (DIC) microscopy of control (green) and patient (red) T cells
while migrating through transwell pores (orange arrowheads) toward CXCL12. Hoechst, blue. Representative of three patients and three controls from
three experiments. (C) Percentage of time T cells (six controls, three patients, from three experiments) spent elongated in 0.2% agarose gel matrices.
(D) Proportions of T cells (eight controls, four patients, from three experiments) elongated during migration on 2D ICAM-coated plates or in 3D collagen
matrices. (E) Proportions of DOCK8-deficient T cells elongated after migration on collagen-coated slides (2D) or in collagen gels (3D), untreated or with
antiintegrin 1 blocking antibodies. Means are shown for three patients and three controls tested under each condition from three experiments. Two-
way ANOVA was performed to compare treatment with or without antibody. (F) Similar to E except that one patient and one control were tested after
transfection of nonspecific or ITGB1 siRNA. Median fluorescence intensities of CD29 for control cells were 6,386 (NS siRNA) and 2,019 (ITGB1 siRNA), and
for patient cells were 6,363 (NS siRNA) and 1,396 (ITGB1 siRNA). Bars, 10 m. A, C, and D show means SD. Linear mixed effects modeling was per-
formed for A, unpaired two-tailed Students t test for C and D, and two-way ANOVA to compare treatment with or without antibody for E. Statistical
significance indicated by ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

infected epicutaneously with HSV, and the donor T cells were
visualized in the skin using intravital two-photon microscopy
(Gebhardt et al., 2011). In contrast to normal T cells, respond-
ing Dock8-deficient T cells showed increased elongation and
fragmentation as they migrated within the combined epider-
mal and dermal layers, indicating that DOCK8 regulates cell
shape integrity in vivo (Fig. 9, D and E; and Video 4).
DOCK8 regulates skin tissueresident memory cell
formation for protection against viral infection
During HSV infection, control of replicating virus requires a
high density of CD8 T cells, which migrate into the epidermis
and develop into tissue-resident memory CD8 T cells (TRM
cells; Gebhardt et al., 2009; Zhu et al., 2013). These cells per-
sist indefinitely after acute inflammation resolves within the
epidermis, where they continuously migrate to protect against
viral reinfection or emergence from latent infection in the skin
(Gebhardt et al., 2011; Ariotti et al., 2012; Jiang et al., 2012;
Mackay et al., 2012; Mueller et al., 2013; Zaid et al., 2014).
In the epidermis, TRM cells adopt a highly ramified cell shape
that is defined by the constraints of the tissue through which
they migrate (Zaid et al., 2014). Thus, we hypothesized that
cytothripsis, associated with defective shape integrity, could
impair the formation or survival of epidermal TRM. This would
decrease the effective virus-specific T cell concentration below
that needed for viral clearance, despite substantial remaining
cytotoxic activity and cytokine production during the immune
response (Zhang et al., 2009; Lambe et al., 2011; Randall et al.,
2011). Other infected organs and normal secondary lymphoid
tissue, in contrast, would offer a less confined environment
for cell migration and reduce these damaging effects on anti-
pathogen immune cells. To test this, normal mice were trans-
ferred with equal mixtures of Dock8-deficient and control
WT T cells, both also expressing the same transgenic TCR that
recognizes an MHC class Irestricted immunodominant HSV
peptide (Mueller et al., 2002). After epicutaneous infection,
Dock8-deficient effector T cells expanded and accumulated
in secondary lymphoid organs and skin with only slightly re-
duced efficiencies as compared with control T cells (Fig. 10 A).
Consistent with previous reports that Dock8-deficient CD8
T cells had reduced long-term survival (Lambe et al., 2011;
Randall et al., 2011), these cells were disproportionately

JEM Vol. 211, No. 13 2555


decreased in the spleens as infection resolved and immuno-
logical memory developed (Fig. 10, B and C). However, the
survival defect was markedly more pronounced in the skin,
where up to 180 times more WT than Dock8-deficient mem-
ory T cells were recovered after 1 mo (Fig. 10 B). When we
examined the skin in these mice by intravital two-photon
microscopy, Dock8-deficient T cells were initially rare but
became largely undetectable by 1 mo after infection, despite
normal numbers of control T cells (Fig. 10 D and Video 5). This
was primarily due to decreased numbers of CD69hi CD103+
Dock8-deficient TRM, which failed to survive in the skin after
HSV infection (Fig. 10 E).
To analyze the formation of CD103+CD8+ TRM cells
in the skin, we cotransferred in vitro activated effector CD8
2556
Figure 7. DOCK8 signals through CDC42
and PAK to maintain shape integrity.
(A) Top: Proportions of T cells elongated during
migration in collagen gel matrices. Normal
human T cells were transfected with nonspe-
cific (NS), DOCK8, CDC42, or RAC1/2 siRNA.
Bottom: representative nuclear morphology of
these cells migrating in collagen gels. BF,
brightfield. DAPI, blue. (B) Proportions of dead
or dying cells were quantitated by flow cyto-
metric analysis of Annexin V and PI staining,
after cells from A were allowed to migrate in
collagen gels or in medium. (C) Knockdown
efficiencies for A and B. Quantitative real-time
RT-PCR to measure transcript levels of DOCK8,
CDC42, RAC1, and RAC2, normalized to -actin.
Knockdown efficiencies of these genes were
calculated as normalized levels in T cells trans-
fected with indicated siRNA, divided by nor-
malized levels in T cells transfected with
nonspecific siRNA. Ratios are means SD of
five independent experiments. (D and E) Pro-
portions elongated within collagen gels of
normal human T cells either treated with
DMSO or the PAK inhibitor IPA3 (D), or trans-
fected with NS or PAK1/2 siRNA (E). (F) Similar
analysis as in B, using cells from E. Represen-
tative immunoblot showing efficiency of
PAK1/2 knockdown. (G) Morphology of T cells
in which WAS gene expression was silenced,
during migration in collagen gel. (H) Propor-
tions of Annexin V+ or PI+ cells, after migrating
in collagen gel matrices or medium. Normal
human T cells were transfected with NS, WAS,
or DOCK8 siRNA. Representative immunoblot
showing WASP knockdown efficiency in human
T cells. Bars, 10 m. A, B, D, E, G, and H are
representative of three independent experi-
ments, and F of five experiments. AF and H
show means SD. One-way ANOVA was per-
formed for A, two-way ANOVA for B, F, and H,
and unpaired two-tailed Students t test for
D and E. Statistical significance indicated by
*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****,
P < 0.0001; ns, nonsignificant.
T cells from Dock8-deficient or control TCR transgenic mice
directly into the skin. This method circumvents any defects in
T cell priming and migration to the skin, allowing direct as-
sessment of the ability of the cells to enter the epidermis and
develop into TRM cells (Mackay et al., 2013). Dock8-deficient
T cells rapidly disappeared in the skin and the few surviving
cells did not up-regulate expression of CD69 and CD103,
which are necessary for TRM formation (Fig. 10, F and G).
Defective TRM survival with severe loss of cells over time re-
sulted in minimal protection against challenge with HSV, as
measured by viral titers in the skin (Fig. 10 H) after transfer
of effector T cells and topical treatment of recipient mice with
the chemical sensitizer 2,4-dinitrofluorobenzene (DNFB) to
generate TRM (Mackay et al., 2012). Together, these results
DOCK8 and cytothripsis in skin viral infections | Zhang et al.

indicate that DOCK8, by regulating cell shape integrity, is
critical in vivo for CD8 T cell persistence, TRM formation, and
antiviral immunity in the skin.
DISCUSSION
While studying the behavior of DOCK8-deficient T cells
and NK cells, we have unexpectedly discovered a new form
of cell death we term cytothripsis for cell shattering. Cyto-
thripsis occurs when shape maintenance fails during lymphocyte
JEM Vol. 211, No. 13
Ar ticle
Figure 8. Abnormal cytoskeletal organization in elongated
cells. In-gel confocal imaging for F-actin (phalloidin, red),
-tubulin (green), and nuclei (DAPI, blue), in normal human
T cells transfected with nonspecific (NS), DOCK8, or CDC42
siRNA, or treated with the PAK1/2 inhibitor IPA3. Representa-
tive of three experiments. Bars, 10 m.
migration. This process can be elicited by the physical prop-
erties of tissues through which the cells migrate, and it is
characterized by cell elongation and nuclear deformation dur-
ing prolonged migration through confined spaces.The result-
ing mechanical forces experienced by the cell likely cause breaks
in the plasma membrane and nucleus that lead to catastrophic
cell death without inducing any biochemical markers of apop-
tosis. Cytothripsis can impair effector T cell and NK cell func-
tions by limiting their effective motility and decreasing their
Figure 9. Defective cell integrity occurs
during response to viral skin infections.
(A) Representative skin disease, 7 d after
epicutaneous HSV infection of WT or Dock8-
deficient (KO) mice. Blue arrowheads, primary
infection sites. Red arrowhead, blistering
along infected dermatome. Orange arrow-
head, blistering with skin necrosis. (B) Daily
scoring of disease severity after acute HSV
infection of WT (black) or KO (red) mice.
(C) Kaplan-Meier survival curves for the mice
from (B). (D) Two-photon imaging of Dock8-
deficient cpm (green) or Dock8-replete T cells
from WT gBT-I mice (red) migrating in the
dermis, 7 d after adoptive cotransfer into WT
recipient mice and HSV infection. White
arrow, fragmentation. Frames show time
elapsed (minutes). (E) Proportions elongated
of the cotransferred T cells in D. Bars, 15 m.
B shows means SEM, with a total of 10 or
14 mice per group from two experiments.
E shows means SD, where data were pooled
from six mice from two experiments. Wilcoxon
matched-pairs signed rank sum test (two-tailed)
was performed for B, log-rank (Mantel-Cox)
test for C, unpaired two-tailed Students
t test for E. Statistical significance indicated
by *, P < 0.05; ***, P < 0.001.
2557

and viral titers measured on day 5. Bar, 100 m. AC and EH show means SD. For A, data were pooled from four experiments with a total of 20 mice
per group. For B, C, and E, data were pooled from four experiments for the day 7 and 31 time points and two experiments for the day 14 time point, with
a total of 1020 mice per time point. F and G are representative of three experiments with seven mice per group. For H, data are from three experiments
with a total of 1315 mice per group. Two-way ANOVA was performed for AC and E, unpaired two-tailed Students t test for F and G, and one-way
ANOVA for H. Statistical significance indicated by **, P < 0.01; ***, P < 0.001; ****, P < 0.001; ns, nonsignificant.
capacity to reach virally infected cells within the tissue in a
viable state. Skin is among the densest tissues within the body
and therefore most likely to elicit cytothripsis, given that
both epidermis and dermis have many highly confined spaces.
Although collagen content is a main determinant of tissue
density in the dermis (Lowry et al., 1941), other environmen-
tal constraints, such as the cellular density and tight junctions
in the epidermis, likely also define the migratory capacity of
leukocytes in tissues. Tissues of intermediate density, such as
the walls of large arteries, may also elicit cytothripsis, thereby
contributing to the vasculopathy associated with local virus
reactivation that has been documented in some DOCK8-
deficient patients (Sabry et al., 2014; unpublished data).
Maintenance of lymphocyte shape integrity, which pre-
vents cytothripsis, is regulated by DOCK8 which, together
with CDC42 and PAK, coordinates cytoskeletal structures
during cell migration. The demand for precise coordination
of cytoskeletal structures is greatest in cells when they ma-
neuver through highly confined spaces, as compared with open
spaces. In lymphocytes, coordination of cytoskeletal struc-
tures is also required during immune synapse formation for
cytotoxicity, directed cytokine secretion, assembly of signaling
platforms for activation and proliferation, and cell division
(Dustin, 2008). External signals determine which of many
2558
Figure 10. Dock8 regulates shape integ-
rity and survival of TRM cells in vivo to
control viral skin disease. (A) Numbers of
WT and Dock8-deficient HSV-specific gBT-I T
cells in the spleen, brachial LNs and skin, 7 d
after adoptive transfer (1:1 mixture) into WT
recipient mice and HSV infection. (B) Relative
numbers of WT gBT-I and Dock8-deficient
cpm gBT-I T cells in spleen and infected skin,
quantified by flow cytometry at the indicated
times after infection. (C) Numbers of WT and
Dock8-deficient gBT-I T cells in the spleen at
various times after skin infection. (D) Two-
photon imaging of skin in C on day 17 after
infection. White arrows, Dock8-deficient cells.
(E) Numbers of WT and Dock8-deficient gBT-I
T cells in skin (CD103+ CD69+ TRM) in C.
(F) Numbers of WT and Dock8-deficient skin
(CD103+ CD69+) TRM, at 23 d after intradermal
cotransfer of in vitroactivated CD8 T cells
into WT recipient mice. (G) Proportions of
CD103+ CD69+ gBT-I TRM that were WT or
Dock8/ in skin in F. (H) Impaired control of
HSV skin infection by virus-specific memory
cells. WT mice received no cells (control) or
in vitro activated effector CD8 T cells from WT
gBT-I or Dock8-deficient cpm gBT-I mice.
After topical treatment with DNFB, mice were
infected at the same site 30 d later with HSV
GEFs become stimulated to activate CDC42, as well as which
effectors downstream of CDC42 become selectively acti-
vated to carry out the different shape rearrangements for
these cellular functions (Sinha and Yang, 2008). This could
explain why DOCK8- and WAS-deficient cells differ in their
migration behavior, and why DOCK8 could contribute not
only to the striking migration defects we have now observed
but also to the lymphopenia and other functional defects that
have been reported in DOCK8-deficient lymphocytes.
Dock8-deficient T cells were previously reported to ex-
hibit a long-term survival defect, as shown by their competi-
tive disadvantage after adoptive transfers of mixtures of mutant
and WT cells (Lambe et al., 2011; Randall et al., 2011). Fur-
thermore, fewer memory CD8 T cells persisted in Dock8-
deficient mice after experimental influenza virus infections
(Lambe et al., 2011; Randall et al., 2011). Our results during
experimental HSV skin infections also indicated that Dock8
mutant T cells are at a competitive disadvantage. However, it
is unlikely that they would survive better in a noncompeti-
tive situation, or at least not sufficiently to restore function,
given that transfer separately of either WT or Dock8 mutant
HSV-specific T cells, followed by challenge of mice after es-
tablishment of memory, conferred no protection in terms of
viral replication. Interestingly, mice conditionally deficient in
DOCK8 and cytothripsis in skin viral infections | Zhang et al.

Cdc42 also showed increased ex vivo expression of apoptotic
markers in their peripheral T cells, which could be rescued in
vivo by overexpressing PAK1 (Guo et al., 2010). Although
the decreased survival was attributed to small decreases in
IL-7 receptor expression for survival signals (Guo et al., 2010;
Randall et al., 2011), our results suggest another mechanism
whereby survival depends not only upon access to survival
signals (unpublished data) but also whether the cell can with-
stand physical stresses as it migrates through the body. Such
stress may have a cumulative effect on trafficking T cells that
is pronounced in certain tissues, such as the skin.
The different migratory patterns of T cells allow them to
perform their important immunosurveillance functions of pre-
venting reinfection or controlling viral reactivation through-
out the body (Mueller et al., 2013). To accomplish this, naive
and central memory T cells continually migrate between blood
and secondary lymphoid organs, whereas effector memory
T cells also migrate through peripheral tissues. When they
circulate through skin, effector memory T cells can be found
moving rapidly through the dermis en route to draining LNs.
In contrast, CD8 memory T cells in the skin do not recircu-
late throughout the body but instead remain indefinitely
within the epidermis where they continuously move. TRM
cells also form in other nonlymphoid tissues in response to
infections, including the intestines, reproductive tract, and lungs,
where they are predominantly associated with epithelial
layers (Mueller et al., 2013). Although the ability of Dock8-
deficient T cells to form TRM populations in those tissues has
yet to be examined, the unique microanatomy of those tissues
may differ from the skin in having less confinement, which
could facilitate some degree of TRM generation. This would
help explain the disproportionate number and severity of viral
skin infections in DOCK8-deficient patients. Nevertheless,
reduced TRM in the lungs or mucosal surfaces may contribute
to the recurrent sinopulmonary infections and chronic ano-
genital viral infections in DOCK8-deficient patients. More-
over, even during acute infections, T cells may be subjected
to physical stresses from the highly confined skin environ-
ment. This could lead to the increased morbidity and mortal-
ity we saw during epicutaneous HSV infections, in contrast
to the normal morbidity previously seen during intranasally
inoculated influenza virus infections of Dock8-deficient mice
(Lambe et al., 2011).
Collectively, our data are highly suggestive that the ab-
normal elongation phenotype is responsible for T cell death
in vivo and that this process prevents TRM formation during
viral infections. However, other explanations, including im-
paired migration and proliferation or alternative mechanisms
of cell death, remain possible. These appear less likely given
our results showing normal chemotaxis, impaired TRM for-
mation even when activated T cells were directly transferred
to the skin, and normal proliferation and activation of T cells
from Dock8-deficient mice (Lambe et al., 2011). Nevertheless,
other immunodeficiencies demonstrate that additional mecha-
nisms exist to control viral skin infections. For example, the
WHIM syndrome, GATA2 deficiency, or the Wiskott-Aldrich
JEM Vol. 211, No. 13
Ar ticle
syndrome, which result from impaired chemotaxis, loss of
peripheral NK cell and dendritic cell differentiation, and/or
defective actin polymerization, can also be associated with
viral skin infections, although the skin infections tend to be
of narrower spectrum or later onset than DOCK8 deficiency
(Sullivan et al., 1994; Imai et al., 2004; Dotta et al., 2011;
Sanal et al., 2012; Spinner et al., 2014). Interestingly, in the
Wiskott-Aldrich syndrome, the defective actin polymeriza-
tion affects many T cell functions including thymopoiesis,
chemotaxis, activation, proliferation, cytokine production,
and cytotoxicity (Zhang et al., 1999; Snapper et al., 2005;
De Meester et al., 2010; Lang et al., 2013). However, when
compared with DOCK8 deficiency, the lack of abnormal elon-
gation and cytothripsis and less frequent occurrence of recur-
rent viral skin infections in Wiskott-Aldrich syndrome patients
(Sullivan et al., 1994) support the concept that lymphocyte
shape integrity contributes to normal protection against viral
skin infections.
In summary, our work has now revealed that the ability to
maintain cell shape integrity is a critical determinant of im-
mune function in rapidly motile cells such as lymphocytes.
Although we have focused on the role of lymphocyte cell shape
integrity in antiviral immunity, our findings are likely to have
general relevance for skin immunity. For example, DOCK8-
deficient patients also often have bacterial or fungal skin infec-
tions, and are at increased risk of developing skin cancers. We
speculate that these conditions result from locally impaired
defense against those pathogens, and possibly impaired tumor
surveillance, when lymphocytes undergo cytothripsis as they
navigate to lesions in skin tissues. Dock8-deficient dendritic
cells also show more globally impaired interstitial migration
that might further compromise skin immunity (Harada et al.,
2012). Maintenance of lymphocyte shape integrity may also
become a factor when cells repeatedly traverse less dense tis-
sues such as blood vessel walls or lymphoid tissues. Because
many T cell populations recirculate between blood, lymphat-
ics, and other tissues, our results may mechanistically explain
the milder systemic defect in peripheral CD8 memory T cell
survival and attrition of naive T cell numbers in DOCK8-
deficient humans or mice (Lambe et al., 2011; Randall et al.,
2011). Finally, our results suggest a more general conclusion
that loss of shape integrity during the navigation of nonim-
mune cells in other migratory processes, such as during em-
bryonic development, could contribute to human disease.
MATERIALS AND METHODS
Patients. Whole blood and leukapheresis samples were obtained from
mutation- and immunoblot-confirmed DOCK8-deficient patients, their rela-
tives, or paid healthy volunteers. Only those patients without somatic rever-
sion or at most 25% of revertant T cells (Jing et al., 2014) were used for the
studies described here. These individuals gave written informed consent to
participate in research protocols approved by National Institutes of Health
(NIH) Institutional Review Boards. Buffy coat cells, which were by-products
of volunteer-donor blood units, and human foreskin tissues (gift from C. Yee,
National Cancer Institute, Bethesda, MD), which were discarded after rou-
tine newborn circumcisions, were distributed in an anonymized manner.
These were exempted from need for informed consent and Institutional
2559
Review Board review, as determined by the NIH Office of Human Subjects
Research Protection.
Mice. Mice were bred and used under animal study protocols approved by
the NIAID Animal Care Use Committee, UK Home Office License, and
the University of Melbourne Animal Ethics Committee. Dock8-deficient
cpm/cpm (cpm) mutant mice, generated by ENU mutagenesis from a pure
C57BL/6 background, gBT-I TCR transgenic mice recognizing an MHC
I-restricted HSV-1 gB epitope, and Actb-DsRed transgenic mice were pre-
viously described (Mueller et al., 2002; Vintersten et al., 2004; Randall et al.,
2009). To obtain Dock8-deficient GFP-expressing mice (GFP-cpm) for cell
tracking experiments, cpm mice were crossed to UBI-GFP transgenic mice
on a C56BL/6 background (The Jackson Laboratory; Schaefer et al., 2001).
Mice were further crossed with gBT-I TCR transgenic mice to enable as-
sessments of virus-specific T cells. WT gBT-I TCR transgenic mice were
crossed to Actb-DsRed transgenic mice for use as controls. In some experi-
ments, Dock8-deficient KO mice were used instead of cpm mice. KO mice
were generated from targeted deletion of exons 9 and 10 in pure C57BL/6
ES cells. WT control C57BL/6 mice were purchased from Taconic or The
Jackson Laboratory. Mice were sacrificed by carbon dioxide asphyxiation or
cervical dislocation for harvest of LNs and spleens.
Cell isolation for in vitro experiments. Human peripheral blood mono-
nuclear cells (PBMCs) were isolated from peripheral blood products by
Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare). T cells
were purified from PBMC by negative selection using human panT cell
isolation kit II (Miltenyi Biotec). In some samples, eosinophils were further
depleted using human CD15 microbeads (Miltenyi Biotec). T cells were ac-
tivated using beads coated with anti-CD2, -CD3, and -CD28 antibodies
provided as a T cell activation/expansion kit (Miltenyi Biotec), using a bead
to cell ratio of 1. Beads were removed on the fourth day after activation, and
cells were expanded in 100 U/ml of recombinant human IL-2 (Aldesleukin;
Prometheus). Activated T cells were cultured for a total of 445 d, when
they acquired an activated effector/memory phenotype, as reflected by loss
of CD45RA and CCR7 surface marker expression, that was comparable be-
tween patients and healthy normal controls. The proportions of revertant
cells were determined in one patient, which appeared stable or mildly in-
creased, ranging from 18% on day 2, 14% on day 8, and 26% on day 21 dur-
ing culture. Human NK cells were purified by positive selection using CD56
microbeads (Miltenyi Biotec) and expanded in 100 U/ml of recombinant
human IL-2 for 68 d. Human T cells and NK cells were typically >90 and
>85% purity, respectively, when used in experiments.
Mouse LNs and spleens were minced into single cell suspensions and
passed through a cell strainer. Splenocytes were also treated with ACK lysing
buffer (Quality Biological). Mouse LN T cells or splenic NK cells were puri-
fied by negative selection, using mouse panT cell or NK cell isolation kits II
(Miltenyi Biotec). Plates were coated overnight with anti-CD3 and anti-CD28
antibodies (BD) at 3 g/ml in PBS to which purified panT cells were added.
After activation for the first 3 d, T cells were expanded in 200 U/ml of re-
combinant human IL-2. Purified mouse T cells were cultured for a total of
57 d. Purified mouse NK cells were activated and expanded with 1,000 U/ml
of recombinant human IL-2 for 67 d. Mouse T cells and NK cells were
typically >90% purity when used in experiments.
Human and mouse cells were grown in complete medium, consisting of
RPMI medium supplemented with 10% FBS, l-glutamine, penicillin, strep-
tomycin, and recombinant human IL-2. Unless otherwise indicated, cells
were kept in the presence of IL-2 throughout subsequent experiments, in-
cluding within 3D matrices.
Lymphocyte immunophenotyping. Standard flow cytometry methods
were used to evaluate cell surface marker expression using various combina-
tions of FITC, PE, PE-Cy5, PE-Cy7, APC, APC-Cy7conjugated anti-CD3,
anti-CD4, anti-CD16, anti-CD25, anti-CD29, anti-CD45RA, anti-CD56, and
PE anti-CXCR4 antibodies (Life Technologies; the rest of the antibodies
are from BD). CFSE dilution assays were performed as previously described
2560
(Zhang et al., 2009). Samples were acquired on a FACSCanto instrument
using FACSDiva software (BD). Analyses were performed using the FlowJo
software version 9 and higher (Tree Star).
Migration studies in human foreskin tissues. Human foreskin tissues
discarded after routine newborn circumcisions were stored in HBSS at 4C
for 37 d before use. Tissues were warmed to 37C overnight in PBS sup-
plemented with 10% FBS, cut into 5 5 mm pieces, and the epidermis
carefully peeled off using a small forceps. T cells, labeled with 0.5 M Cell-
Tracker Green CMFDA (5-chloromethylfluorescein diacetate; Life Tech-
nologies), were dropped onto the exposed dermis and allowed to migrate
into the tissues at 37C for 3 h. Cells remaining on the dermal surface were
gently rinsed away, and tissues immersed in complete medium within
Lab-Tek II 8-well chambered coverglasses (Nunc). Live cell imaging was per-
formed at 37C in the presence of 5% CO2, using a TCS SP5 laser scanning
confocal microscope (Leica) with excitation at 488 nm. Image stacks were
collected with a 4-m vertical step size at a depth of 4060 m. Collagen was
visualized by refractive imaging.
RNA interference. Human T cells, 47 d after initial activation, were nu-
cleofected using the Lonza 96-well shuttle system according to the manufac-
turers optimized protocol. Stealth select prevalidated siRNA against DOCK8,
ITGB1, CDC42, RAC1, RAC2, PAK1, PAK2, WAS, or nonspecific nega-
tive control siRNA were used (Life Technologies). To optimize knockdowns,
cells were nucleofected 2 d in a row, and the dead cells removed by Ficoll-Paque
PLUS gradient centrifugation on the third day. Experiments were performed
and lysates harvested to analyze RNA or protein expression on the fourth
day after nucleofection. For PAK1/2 knockdown, cells were nucleofected
every other day for four times, the dead cells removed by Ficoll-Paque PLUS
gradient centrifugation 1 d later, and used for experiments and evaluation of
knockdown efficiency on the second day after the last nucleofection.
Immunoblotting. Immunoblotting for DOCK8 protein and -actin was
performed as previously described (Zhang et al., 2009). For analysis of other
proteins, cells were lysed in 2% SDS (50 mM Tris HCl, pH 6.8, 10% glycerol)
or 1% NP-40 (1 mM EDTA, 50 mM Hepes, and 150 mM NaCl) containing
complete protease inhibitors (Roche). Protein lysates were separated on Nu-
PAGE Novex 12% or 412% gradient Bis-Tris gels using MOPS SDS or
MES running buffers (Life Technologies), followed by wet or semi-dry trans-
fer. Membranes were blocked with 5% blocking grade nonfat milk (Bio-Rad
Laboratories) or blk-CH (Millipore). Antibodies were to caspase-9 (BD);
caspase-8 (clone C15; gift from L. Zheng, NIAID, NIH, Bethesda, MD); and
PAK1, PAK2, and WASP (Santa Cruz Biotechnology, Inc.).
Quantitative real-time RT-PCR. Total RNA from transfected primary
T cells was isolated using the RNeasy mini kit with DNaseI in-column di-
gestion (QIAGEN). 0.9 g of total RNA was reverse-transcribed with Su-
perscript III first-strand synthesis supermix (Invitrogen). Diluted cDNA was
analyzed by quantitative real-time PCR using Power SYBR Green PCR
master mix on a 7500 Real Time PCR System (Applied Biosystems). Stan-
dard conditions of 40 cycles (95C for 15 s, and 60C for 1 min) were used.
RNA quantity was calculated from the cycle number by using primer-specific
standard curves. Primer sets designed to span exon junctions were as follows:
DOCK8 forward (F), 5-TCAGCCTCTGTGGGTAGACA-3, and reverse
(R), 5-CCGCACAAAGAGATTTTGGA-3; CDC42 F, 5-CGCCCA-
CAACAACACACTTA-3, and R, 5-CCCGGTGGAGAAGCTGAG-3;
RAC1 F, 5-TCTCCAGGAAATGCATTGGT-3, and R, 5-CTGATG-
CAGGCCATCAAGT-3; RAC2 F, 5-TTGCTGTCCACCATCA-
CATT-3, and R, 5-AGACCTGCCTTCTCATCAGC-3; and -actin
F, 5-GTTGTCGACGACGAGCG-3, and R, 5-GCACAGAGCCTC-
GCCTT-3. Expression of each gene was normalized to the -actin house-
keeping gene. Knockdown efficiencies of genes were calculated as normalized
levels in T cells transfected with indicated siRNA, divided by normalized
levels in T cells transfected with nonspecific siRNA.
DOCK8 and cytothripsis in skin viral infections | Zhang et al.

Collagen gel migration assays for morphological analyses. Collagen
solution from bovine skin (Sigma-Aldrich) was mixed with 10 RPMI 1640
medium (Life Technologies) and FBS, to a final concentration of 3.6 mg/ml
(unless otherwise indicated) of collagen, 1 RPMI, and 10% FBS. l-glutamine,
penicillin, streptomycin, and 200 U/ml of recombinant human IL-2 were
also added. The collagen mixture was stored at 4C for no more than 10 d
before use. Cells were then admixed at a final concentration of up to 5
107 cells/ml and the collagen gel matrix polymerized at 37C for at least
1 h in Lab-Tek II 8-well chambered coverglasses (Nunc). In initial experi-
ments, addition to bovine skin collagen of fibronectin at 6 g/ml and mouse
laminin at 2.5 g/ml (both from Life Technologies), or substitution of Cul-
trex rat tail collagen (Trevigen) for bovine skin collagen, made no difference.
Therefore, bovine collagen alone was used in all subsequent experiments.
Where indicated, small molecule inhibitors, or DMSO used as a vehicle
control, were mixed with the collagen matrix at 4C, before addition of cells
and polymerization of gels. Minimal doses that exerted effects while avoiding
nonspecific toxicity were used. In some experiments, Z-Val-Ala-Asp (OMe)-
FMK (zVAD) (MP Biomedicals) was added at 40 or 80 M to gels for 1218 h
to inhibit caspase activation. The class I PAK inhibitor IPA3 (Sigma-
Aldrich) was used to pretreat cells at 5 m for 30 min but was not added to
gels to minimize cell toxicity. In other experiments, anti1-integrin (ITGB1
or CD29) antibodies (clone P5D2), which have been shown to block adhe-
sion to collagen (Blaschke et al., 2002; Mukhopadhyay et al., 2004), were
added to the gels at a final concentration of 20 g/ml (R&D Systems).
After polymerization of gels, cells were allowed to migrate within the
gels at 37C in the presence of humidified 5% CO2. Migrating cells were
visualized by diffusion interference contrast (DIC) microscopy using an
AF6000 LX microscope (Leica) on a motorized stage, with either a 20 dry
objective lens or a 63 glycerol immersion objective lens. Images were ac-
quired at 30-s intervals. Post-capture analysis was conducted with Imaris 7.0
software (Bitplane). Length was determined by measuring the distance be-
tween cell front and uropod. Width was determined by measuring the middle
point across the cell body. Cells were defined as abnormally elongated if cell
length was 8 the cell width at its midpoint. Abnormal elongation was re-
ported either as percentage of time elongated or percentage of cells elongated.
To calculate percentage of time elongated, at least 30 min of captured live
images were analyzed. For each sample, cells were numbered and 2030 cells
were randomly chosen using an online random number generator. The
lengths and widths of these cells were measured in each frame. The percent-
age of time each individual cell spent abnormally elongated was calculated,
which in turn was used to calculate the mean value for the sampled cells from
each patient. To calculate percentage of cells elongated, at least 3 h of cap-
tured live images were analyzed. Four time points that divided up the total
time imaged into equal time intervals, and three z-steps associated with each time
point, were selected for analysis. The lengths and widths of all the cells in the
view at these four time points were measured. The average proportion of
cells that were abnormally elongated was calculated from the sampled time
points from each patient. Approximately 150300 cells were analyzed by this
latter method. The percentage of cells with elongated nucleus was calculated
similar to the percent of cells elongated, except that Hoechst dye 33342 was
used to stain nuclei and nuclei were classified as elongated as determined by
binary scoring of blinded samples.
Immunofluorescence. T cells migrating within the collagen gel matrix
were fixed with prewarmed 4% paraformaldehyde (Electron Microscopy Sci-
ences) in PBS. The collagen gel containing the cells was washed in PBS, per-
meabilized with 0.5% Triton X-100, blocked with 2% bovine serum albumin
(Sigma-Aldrich) in 0.1% Triton X-100, and incubated with primary antibod-
ies for 1 h at 4C. Anti-TubulinAlexa Fluor 488 (Life Technologies) was
used with anti-pericentrin antibody (Abcam), followed by Alexa Fluor 647
conjugated antirabbit secondary antibody (Life Technologies). Hoechst dye
33342 at 1 g/ml (Life Technologies) was also used. Stained gels were kept
in PBS at 4C for no longer than 24 h before image capture. Imaging was
performed on a Leica SP8 or SP5 white light laser confocal microscope with
a 63 glycerol immersion objective lens, using an image stack vertical step
JEM Vol. 211, No. 13
Ar ticle
setting of 0.150.2 m. Where indicated, after staining gels were physically
compressed to facilitate simultaneous visualization of all vertical stacks.
Cell cycle analysis. Cell cycle analysis was performed according to standard
protocols (Darzynkiewicz and Huang, 2004). In brief, after fixation in 70%
ethanol on ice for 2 h, cells were stained with 20 g/ml of PI in PBS contain-
ing 0.1% Triton-100 and 0.2 mg/ml DNase-free RNase A (QIAGEN) at
37C for 15 min. DNA content was acquired on a FACSCanto using a linear
fluorescence amplification scale. Area versus width was used to gate out dou-
blets. To calculate cell cycle, data were analyzed by using the Dean-Jett-Fox
model on the Cell Cycle analysis platform of FlowJo.
Cell cycle blockade. Patient cells that had been expanded in culture were
enriched for viable cells by Ficoll-Paque PLUS density gradient centrifuga-
tion before use in experiments. Cells were treated for 48 h with aphidicolin
at 2 g/ml (Sigma-Aldrich) or were treated with DMSO. Cells were kept in
the presence or absence of aphidicolin when mixed into the collagen mixture
and the gel allowed to polymerize. After migration for 24 h in the gel, cells
were visualized by DIC microscopy to calculate the percent of cells elon-
gated, as described above. Alternatively, cells were fixed in the gel using 2%
paraformaldehyde and stained with Hoechst dye 33342. For abnormally
elongated cells, the nuclear length was measured as a proportion of the cells
total length. From 13 to 38 elongated cells were analyzed per patient. Cell
cycle analysis performed on cells recovered from the gel according to colla-
genase treatment showed that aphidicolin treatment decreased the percentage
of cells in S/G2/M from 17.0 10.5 and 13.2 4.0 in pretreated controls
and patients, respectively, to 1.4 0.6 and 1.0 0.5 in post-treated controls
and patients, respectively (mean SD).
Chemotaxis assays. 3D chemotaxis assays were performed as described
using a custom-fabricated device, with modifications (Sixt and Lmmermann,
2011). Activated T cells, resuspended in 3.6 mg/ml collagen, as described
above, were loaded into the migration chamber. After polymerization of the
gel matrix containing the cells, additional collagen gel, containing recombi-
nant human CXCL12 (SDF-1; PeproTech) at a final concentration of
400 ng/ml, was applied as a second layer within the migration chamber.
Cells were visualized over 30 min as they migrated toward the chemokine
gradient at 37C in the presence of humidified CO2. Time-lapse DIC im-
ages of migrating cells were acquired using a microscope (AF6000 LX; Leica)
at intervals of 30 s. Cell images were loaded into Imaris 7.0, and individual
cells were tracked using the Track Spots feature in the DIC channel. The
estimated diameter of the cells was 8 m, and a Quality Threshold for the
spots was typically around 100. Tracks of the individual cell spots were as-
sembled using the autoregression feature. Tracks shorter than 30 time points,
or in which the cell displaced less than 5 m, were excluded. The path trav-
eled was used to calculate total displacement, displacement to the chemo-
kine, speed, and directional velocity toward the chemokine. A total of 60326
cells were analyzed to obtain means per sample. For statistical analysis, the
values from DOCK8-deficient cells from patients and knockdowns were
combined and compared with control cells and nonspecific siRNA knock-
downs, using the unpaired Students t test.
Live imaging of dying cells. In initial experiments to assess cell death,
standard collagen gel matrices were set up as described above in Collagen gel
migration assays for morphological analyses. DIC images were acquired at
2-min intervals for 21 h. PI was added during the last 4.5 h and allowed to
diffuse into the gel, during which time simultaneous DIC and fluorescence
images were acquired at 510 min intervals.
In subsequent experiments, mini-gels were used to track the fate of
cells for 16 h. 10 l of collagen gel, which contained 104 cells, was cast into
a custom-partitioned segment of a high culture-insert StemCell, ibiTreat-coated,
35 mm -Dish (ibidi). After polymerization, all cells within the mini-gel
were visualized at 37C in the presence of humidified 5% CO2 by DIC
microscopy. A microscope (AF6000 LX) with 20 dry objective lens and
motorized stage with Tiling function was used to scan through the entire
2561
mini-gel every 2 min. Image stacks were acquired using a Roper Coolsnap
camera, using the following settings: 2 2 binning, 1 3 tiling, 8 m verti-
cal step, 300 m vertical depth. After data collection, tiles were assembled
using LAS EZ software (Leica) and analyzed manually using Imaris software
as described above. Cells were classified as those that had died or remained
alive at the end of the experiment. The amount of time each cell spent ab-
normally elongated was calculated from the cell length and width measure-
ments at each time frame. Elongation episodes were also classified as those
immediately preceding cell fragmentation and those that did not. The dura-
tion of each elongation episode was also measured. 20 cells, chosen ran-
domly at the beginning of the experiment, were analyzed per sample.
Quantitation of cell death. Cells were mixed into collagen gel matrices
containing varying concentrations of collagen, or in complete medium, which
were both supplemented with 200 U/ml of recombinant human IL-2. At
the indicated times, cells were recovered by treating samples with 1 mg/ml
of collagenase (Sigma-Aldrich) with 2.5 mM CaCl2 at 37C for 1 h. To stain
for cell death markers, APC-conjugated Annexin V (BD) was incubated at
4C for 15 min with the recovered cells. PI was added right before acquisi-
tion for 30 s at high speed on the FACSCanto. The numbers of Annexin V+,
PI+, or live (Annexin V, PI) cells were counted using FlowJo analysis soft-
ware. These numbers were used to calculate the percentage of cells that were
dead or dying cells, as defined by Annexin V+ and/or PI+.
Scanning electron microscopy. After allowing 105 cells to migrate within
the collagen gel matrix, cells were isolated by treatment with collagenase as
described above and washed with PBS. Recovered cells were resuspended in
100 l of 0.1 M sodium cacodylate buffer and 50 l of suspensions were set-
tled on silicon chips (Ted Pella, Inc.) for 20 min. The silicon chips were then
fixed and stored at 4C for up to 20 h in fixative containing 2.5% glutaralde-
hyde and 0.1 M sodium cacodylate buffer in PBS, pH 7.4. Samples were
treated for 1 h with 0.5% osmium tetroxide and 0.8% potassium ferricyanide,
1 h with 1% tannic acid, and then overnight at 4C with 1% uranyl acetate.
After a graded series of ethanol dehydration steps, the silicon chips were crit-
ical point dried (cpd 030; Bal-Tec), mounted on aluminum studs, and coated
with 70 iridium in an IBS/e sputter coater (South Bay Technologies).
Digital images were acquired at 5 kV using a SU-8000 field emission scan-
ning electron microscope (Hitachi High Technologies).
Caspase-3 staining. For intracellular flow cytometric detection of acti-
vated caspase-3, T cells were allowed to migrate within the collagen gel ma-
trix, or in medium, for up to 20 h. Apoptosis was induced in control cells by
allowing them to migrate for 6 h within the collagen gel matrix, to which
anti-FAS (clone APO-1-3) antibodies (Enzo Life Sciences) and Protein A
(Sigma-Aldrich) were incorporated at 200 ng/ml. Cells were recovered
from gel or medium after collagenase digestion, as described above. Cells
were then fixed and permeabilized using the Cytofix/Cytoperm Fixation/
Permeabilization kit (BD). Cells were incubated with a 1:10 dilution of
PE-conjugated antiactive-caspase-3 antibody (BD) in Perm/Wash buffer
for 30 min at 4C. Stained cells were acquired on a FACSCanto, and per-
centage of positive cells was calculated using FlowJo analysis software.
Mitochondrial membrane permeability staining. Cells were loaded
with 0.5 g/ml of JC-1 (Life Technologies) at 37C for 30 min and then
washed with complete medium. Loaded cells were incorporated within a
collagen gel matrix as described above, and allowed to migrate within the gel
for 6 h. In some cases, apoptosis was induced in control cells by incubating
JC-1loaded cells with 200 ng/ml of anti-FAS (clone APO-1-3) antibodies
and 200 ng/ml of Protein A for 20 min, before incorporating cells within the
collagen gel matrix. Live cell imaging, at 37C and in the presence of 5%
CO2, was performed using a white light laser confocal microscope (Leica),
with excitation wavelength of 488 nm and an image stack vertical step setting
of 0.2 m. Loss of orange-red staining of J aggregates, with continued green
staining of J monomers, was indicative of mitochondrial depolarization.
2562
Transwell assays. Polycarbonate Transwell inserts containing 5-m mem-
brane pores were used in 24-well plates (Corning). Recombinant human
CXCL12 (SDF-1) at 200 ng/ml (Life Technologies) was added to the lower
compartment. Control and patient T cells were loaded with 0.5 M of Cell-
Tracker Green CMFDA or CellTracker Red CMPTX (Life Technologies),
and dye swaps were also performed. Cells were washed once with com-
plete medium before placing a total of 5 104 cells in the upper compart-
ment. After incubating at 37C, in humidified 5% CO2, for 12 h, the
insert was removed, fixed in 2% paraformaldehyde in PBS for 15 min,
rinsed in PBS, and stained with Hoechst dye 33342. The membrane was
carefully cut off from the insert using a sharp scalpel and mounted onto
glass slides with coverslips using ProLong Gold mounting medium (Life
Technologies). Cells migrating through the membrane pores were imaged
using a TCS SP5 laser-scanning confocal microscope with a 63 glycerol
immersion objective lens.
Agarose gel migration assays. Tissue-culture grade agarose (Sigma-
Aldrich) was heated at a concentration of 2% in RPMI medium. A heating
block was used to keep the mixture warm as it was diluted with prewarmed
complete medium to a final concentration of 0.2% agarose. After equilibra-
tion to 37C, cells were admixed. Gels were cast into Lab-Tek II 8well
chambered coverglasses, solidified at 4C for 10 min, and moved back to
37C in the presence of humidified 5% CO2. After migration within the
agarose gel matrix, live cell imaging and analysis was performed as described
above in Collagen gel migration assays.
2D migration assays. Lab-Tek II 8well chambered coverglasses (Nunc)
were coated with 3 g/ml of recombinant human ICAM-2/Fc chimera
(R&D Systems) overnight at 4C and rinsed with PBS before use. Alterna-
tively, collagen-coated slides were stored at 4C until use (BD). 200 l of
cycling T cells (105/ml) were gently added into each well. After 1 h incuba-
tion at 37C in the presence of humidified 5% CO2, time-lapse DIC micros-
copy and analysis was performed as described above in Collagen gel migration
assays for morphological analyses.
HSV-1 stocks and titers. To generate cell-free virus stocks of the KOS
laboratory-adapted strain of HSV-1 (gift from J. Cohen, NIAID, NIH,
Bethesda, MD), 1.2 107 Vero cells grown in DMEM medium were seeded
1 d earlier to obtain a confluent monolayer. 2 104 pfu of virus was used to
infect the Vero cells. Virus was allowed to replicate while cells were cultured
for 2 d at 37C, in the presence of humidified 5% CO2. To harvest the virus,
the infected cells were collected into the medium using a cell scraper, and
centrifuged briefly at 4C. The pellet was frozen on dry ice and thawed for
three cycles. After centrifugation at 4C, the supernatant was aliquoted and
stored at 80C until use.
Virus stocks were quantitated using a standard plaque assay. Vero cells
were seeded at 5 105 cells per well in 6-well plates, 1 d earlier. An aliquot
of virus stock was thawed on ice and a 10-fold dilution series was made in
medium. In each well, 500 l of the diluted virus was added to a total volume
of 1 ml, and virus allowed to adsorb for 1 h. Cells were overlaid with medium
containing human gamma globulin (Gamunex-C, Talecris) and cultured for
2 d. The medium was removed, and cells were stained for 1 h at room tem-
perature with 1% crystal violet in 10% formaldehyde, 5% acetic acid, and 60%
methanol. After washing with water and air-drying, the plaques in the cell
monolayers were counted. The PFU/ml of the virus stock was calculated as:
(number of plaques in one well) 2 (fold of dilution of virus stock used
for infection).
HSV-1 infections. Mice were infected epicutaneously as previously de-
scribed (Goel et al., 2002; van Lint et al., 2004; Gebhardt et al., 2011). Sex-
matched mice, at 68 wk of age, were used for infections, with four to seven
mice per treatment group in each experiment. In brief, mice were anesthe-
tized with an intraperitoneal injection of 2.5 mg ketamine and 0.4 mg xyla-
zine in sterile PBS, per 20 g of body weight. The fur was clipped and depilated
with Nair in the region of the dorsal left flank. The skin was then abraded
DOCK8 and cytothripsis in skin viral infections | Zhang et al.

using a 1.5 1.5 mm square, 100 grit sand paper (3M), with 10 steady strokes
of manually applied pressure. HSV-1, at 106 pfu in a volume of 5 l, was dropped
onto the abraded skin, and the site of infection covered for 12 h. Disease se-
verity was scored daily according to the following criteria: 0, no symptoms;
1, several isolated blisters close to the original infection site; 2, blisters clus-
tered along a dermatomal distribution to form a continuous line; 3, blisters
merged together with extensive necrosis surrounding the blisters; 4, mouse
found dead or with neurological symptoms requiring euthanasia.
Adoptive transfers of T cells. CD8 T cells were isolated and enriched
from DOCK8-deficient GFP-cpm mice by incubating splenocytes with an
antibody cocktail (Ter119, M5/114, GK1.5, RB6-8C5, M1/70, and F4/80)
and performing negative enrichment with Dynabead magnetic beads (Invit-
rogen). These T cells were adoptively transferred into C57BL/6 mice (3
106 cells) along with 3 103 CD8 T cells from gBT-I.DsRed TCR trans-
genic mice, before skin HSV infection. Naive gBT-I.DsRed or gBT-I.GFP.
cpm T cells isolated from LNs were adoptively transferred via the tail vein.
A total of 5 104 cells were transferred in cotransfer experiments before HSV
infection. In vitrogenerated gBT-I and gBT-I.GFP.cpm effector spleno-
cytes were activated by peptide-pulsed splenocytes as previously described
(Mackay et al., 2012). Such cells were comparably activated by this regi-
men, as reflected by their CD44hi and CD62Llo surface marker expression,
consistent with previous reports that Dock8 mutant cells activate and pro-
liferate normally after TCR stimulation (Lambe et al., 2011). Activated
CD44hi T cells (106) were transferred into recipients by intradermal injec-
tion (five 20-l injections over an area of skin 1 1.5 cm2) with a 30-gauge
needle. For DNFB (Sigma-Aldrich) treatment, mice were shaved and depil-
ated before the application of 15 l of 0.5% DNFB in acetone/oil (4:1) to
a 1-cm2 area of skin. 30 d after DNFB treatment, mice were infected on the
skin with HSV.
Intravital two-photon microscopy. Mice were infected epicutaneously
with HSV-1 and the skin imaged by two-photon microscopy as previously
described (Gebhardt et al., 2011). For intravital two-photon microscopy, mice
were anaesthetized, depilated, and two parallel incisions were made 15 mm
apart along the flank. The skin was separated from the peritoneum, and ad-
hered to an 18-mm-wide piece of 1-mm stainless steel inserted to form a
stable raised platform, attached to a custom-made imaging platform main-
tained at 35C. Images were acquired with an upright LSM710 NLO multi-
photon microscope (Carl Zeiss) with a 20 1.0 NA water immersion
objective. 3D stacks were captured every 1 min for 3060 min. Raw imag-
ing data were processed with Imaris 7 (Biplane). Cell migration through
combined epidermal and dermal layers was analyzed through automatic cell
tracking aided by manual corrections. Only tracks that lasted longer than
5 min were analyzed. For the generation of movies, image sequences ex-
ported from Imaris were composed in After Effects (CS5; Adobe).
Statistical analyses. Figs. 2 (CI), 4 (D and E), 5 (C and F, right), 6 (C and D),
7 (D and E), 9 E, and 10 (F and G): the unpaired two-tailed Students t test
was performed using Prism 6.0 software (GraphPad). Fig. 9 E: 17 movies
from six mice pooled from two experiments were analyzed. In each movie,
all the cells in the frame, numbering at least several dozen, were counted and
the proportion elongated calculated. In the remaining experiments where
individual cells were analyzed, the mean value for the population from each
sample tested was used to represent that individual. P-values are indicated
in the legends. Fig. 3 (AD): the mean values for controls and control NS
siRNA were included in the DOCK8-replete group, and the mean values
for patients and knockdowns were included in the DOCK8-deficient group.
For each parameter, the two groups were compared using the unpaired two-
tailed Students t test. ns, not significant (P 0.4). Figs. 4 C, 5 F (left), 6 E,
7 (B, F, and H), and 10 (AC and E): ordinary two-way ANOVA was per-
formed with correction for multiple comparisons using Prism. P-values are
indicated in the legends. For Fig. 6 E comparisons, p-values were nonsignifi-
cant (P > 0.99). Fig. 5 C: for each individual tested, the time was plotted
against percentage of active caspase-3positive cells. The slopes of the
JEM Vol. 211, No. 13
Ar ticle
individual doseresponse curves were calculated using the nonlinear regres-
sion curve fitting option (straight-line) in Prism. The slopes for controls mi-
grating in gel and patients migrating in gel were 0.015 0.023 and 0.13
0.054, respectively, which indicated little dose-effect correlation for cells mi-
grating in gel. In contrast, in controls induced to undergo apoptosis, the slope
was 11.0 1.4, which indicated a strong dose-effect correlation (mean
SD). Fig. 6 A: a linear mixed effects model was used to compare the effect
of collagen concentration on response of cells from controls versus patients.
There was no dose effect in the control group, and no difference between
controls and patients when dose level was 0. The dose effect at concentra-
tions >0 was different in the controls versus the patients, as indicated by in-
teraction of 4.736 (95% CI of 3.87, 5.61) with p-value <0.0001. Figs. 7 A
and 10 H: ordinary one-way ANOVA was performed with correction for
multiple comparisons, using Prism. P-values are indicated in the legends.
Fig. 9 B: The Wilcoxon matched-pairs signed rank test (two-tailed) was
performed using Prism to compare repeated measurements of clinical scores
associated with individual Dock8-deficient (KO) versus WT mice. The
p-value was 0.001. Fig. 9 C: the log-rank (Mantel-Cox) test for the Kaplan-
Meier survival curve was performed using Prism. *, P = 0.016.
Online supplemental material. Videos 1 and 2 show T cell and NK cell
elongation during migration within collagen gel matrices. Video 3 shows
cytothripsis. Videos 4 and 5 show intravital two-photon microscopy during
viral skin infections. Online supplemental material is available at http://
www.jem.org/cgi/content/full/jem.20141307/DC1.
We thank the following people: for pictures of patient skin and skin histology,
Maria Turner and Stefania Pittaluga; for technical assistance with microscopy
studies, Juraj Kabat and Owen Schwartz; for electron microscopy, Beth Fischer; for
statistical analyses, Jin Qing; for mouse breeding and screening, Gayle Davey and
Melanie Damtsis; for reagents, Fabio Candotti, Jeffrey Cohen, Carole Yee, and Lixin
Zheng; for clinical support, Thomas Dimaggio and Angela Wang; for helpful advice
and/or critical reading of the manuscript, Tim Lmmermann, Michael Lenardo,
Pamela Schwartzberg, Andrew Snow, Li Yu, and Yu Zhang. We also thank the
patients and their families for participating in this study, especially Kelsey Koch, in
whose memory we dedicate this work.
This work was supported by the Intramural Research Program of the NIAID,
NIH, the Australian Research Council (S.N. Mueller), the Medical Research Council
UK (R.J. Cornall), and the Oxford NIHR Biomedical Research Centre (R.J. Cornall).
R.A. Grodick was an HHMI-NIH Research Scholar.
The authors have no conflicting financial interests.
Author contributions: C.G. Dove and Q. Zhang discovered and characterized the
3D migration defect in collagen gel matrices. D.M. Strauss-Albee performed
foreskin migration experiments. Q. Zhang, D.M. Strauss-Albee, and C.G. Dove
discovered and characterized the nuclear deformation abnormality. R.A. Grodick
performed cell cycle blockade and agarose gel experiments. C.G. Dove and
T.E. Lenardo performed chemotaxis experiments. Q. Zhang, J.A. Garcia, and
C.G. Dove discovered and characterized cytothripsis. Q. Zhang and C.G. Dove
performed immunofluorescence experiments. Q. Zhang performed Transwell
and 2D migration and integrin blockade/knockdown experiments. H.M. Murdock,
Q. Zhang, J.A. Garcia, and D.B. Chandler-Brown performed CDC42, RAC, and
PAK knockdown and in-gel immunoblotting experiments. Q. Zhang and J.A.
Garcia performed WAS knockdown experiments. H.M. Murdock performed
small molecule cytoskeletal inhibitor experiments. S.N. Mueller, J.L. Hor,
G. Crawford, and R.J. Cornall generated mouse strains. Q. Zhang and J.N. Mandl
evaluated disease, and J.L. Hor and S.N. Mueller performed intravital two-
photon imaging and TRM studies in HSV-infected mice. A.F. Freeman and H.C. Su
diagnosed patients and provided clinical information and samples. H.F. Matthews
coordinated clinical study protocol and sample collection. H. Jing and Q. Zhang
assisted with identifying patients and processing of samples. H.C. Su and S.N.
Mueller planned and supervised the experimental work and data analyses, and
R.N. Germain provided advice. Q. Zhang and H.C. Su prepared the manuscript. All
authors discussed and revised the manuscript.
Submitted: 11 July 2014
Accepted: 29 October 2014
2563
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DOCK8 and cytothripsis in skin viral infections | Zhang et al.
INSIGHTS

FLUshing in the bathroom

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 gastro-
enteritis-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/CCR9-
dependent 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 regu-
latory 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

ADAPTED FROM AN ILLUSTRATION BY THE AUTHORS

TGF-b, regulatory cells and continuous regenera-
tion 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/ Working model of intestinal injury induced by respiratory influenza virus infection
jem.20140625.

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


 Article

Respiratory influenza virus infection induces

intestinal immune injury via microbiota-
mediated 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
1Instituteof 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 diar-

rhea, 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 oc-
curred, 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 dis-
ease; IEC, intestinal epithelial
cell; i.g., intragastrical(ly); i.n.,
intranasal(ly); SFB, segmented
filamentous bacteria.
Influenza is an infectious respiratory disease af-
fecting 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 influ-
enza patients, such as abdominal pain, nausea,
vomiting, and diarrhea, especially in young chil-
dren (Baden et al., 2009; Shinde et al., 2009;
Dilantika et al., 2010). However, the immune
mechanisms underlying these clinical manifes-
tations 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 essen-
tial in shaping intestinal immune responses dur-
ing both health and disease (Hooper and Gordon,
*J. Wang and F. Li contributed equally to this paper.
2001; Chervonsky, 2009). Distinct components
of commensal bacteria were associated with spe-
cial 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 bac-
teria have been suggested to influence suscepti-
bility 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 in-
testinal disease.
It is well known that the respiratory and intes-
tinal tracts are both mucosal tissues. Over 30 years
ago, John Bienenstock hypothesized that the
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J. Exp. Med. 2014 Vol. 211 No. 12 23972410 2397
www.jem.org/cgi/doi/10.1084/jem.20140625

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 inter-
play 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 understand-
ing 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 com-
ponents 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 in-
duced by influenza infection of the lung but also the interplay
of immune cells between or among different mucosal sites.
2398
Figure 1. Respiratory influenza virus
infection causes lung and intestinal im-
mune 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 un-
formed stool with moderate perianal staining
of the coat; and 3, watery stool with severe
perianal staining of the coat). (E) The pathol-
ogy 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 sec-
tions were stained with H&E. Bars, 100 m.
Data represent three independent experi-
ments 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.
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 grad-
ually 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 intes-
tine 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 infec-
tion 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 possi-
bility 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.

(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 unexpect-
edly observed that influenza infection induced severe immune
injury within the intestine only when the virus infected the re-
spiratory 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 oc-
currence 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 infec-
tion as quantified by both real-time PCR and selective cul-
ture (Fig. 3 A), the numbers of segmented filamentous bacteria
(SFB) and Lactobacillus/Lactococcus decreased after PR8 infec-
tion, whereas the number of Enterobacteriaceae increased; more-
over, 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 micro-
biota (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
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 influ-
enza 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 pathol-
ogy 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 re-
corded 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.
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 intesti-
nal 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 in-
testinal microbiota.
Escherichia coli is an important component of Enterobacteria-
ceae, 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 dif-
ferent 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 re-
duce 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 intesti-
nal 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 intes-
tine 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 Microbiota cause intestine injury during influenza | Wang et al.


suggesting that Th17 cells might be involved in influenza-
induced 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 re-
sults 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 re-
duced body weight loss during PR8 infection (Fig. 4 E). How-
ever, 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 re-
duce in IL-17A/ mice after influenza infection when com-
pared 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 kid-
ney (Fig. 5 D), consistent with previous observations (Esplugues
JEM Vol. 211, No. 12
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 expres-
sions in the lung from WT mice were de-
tected by real-time PCR 6 d after PR8
infection. (E) Body weight of WT and
IL-17A/ mice was monitored after PR8 infec-
tion. (F) Evans blue dye concentration in BALF
from WT and IL-17A/ mice was determined
by spectrophotometer 6 d after PR8 infec-
tion. (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 sec-
tions 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.
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 depen-
dent on Th17 cells.
Because we observed that influenzainduced intestinal im-
mune 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 in-
testine 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 treat-
ment inhibited IL-17A expression in the small intestine dur-
ing PR8 infection (Fig. 5 H). Collectively, these data suggest
that changes in intestinal microbiota induced by influenza in-
fection 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 compo-
sition 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

Given that influenza infection specifically caused immune in-
jury 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 lym-
phocytes into the small intestine as a homing mechanism
(Campbell and Butcher, 2002). Consistent with previous ob-
servations, 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 anti-
CCL25 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
2402
Figure 5. Increased Th17 cells occur in the
small intestine during influenza virus infec-
tion. (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 de-
tected 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 saline-
treated 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.
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 recruit-
ing 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 popula-
tion, 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 al-
tering 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),
Microbiota cause intestine injury during influenza | Wang et al.
 Ar ticle

Figure 6. Anti-CCL25 antibody treatment reduces influenzainduced intestinal immune injury. (A) CCL25 expression in various tissues was de-
tected 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 mes-
enteric 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.

JEM Vol. 211, No. 12 2403

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

2404 Microbiota cause intestine injury during influenza | Wang et al.


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 dehy-
drogenase (ALDH) 1A2 (Yokota et al., 2009). In our study, the
expression of ALDH1A2 in lung increased after influenza infec-
tion (Fig. 6 H), suggesting that the increase of retinoic acid in
lung after influenza infection might be responsible for promot-
ing 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 PR8-
infected 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 in-
fection, 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 mi-
crobiota during influenza infection, we wondered how they
influenced the intestinal microbiota composition and whether
Th17 cells in the small intestine originated from the polariza-
tion 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 ex-
hibited reduced intestinal immune injury, normal IL-17A ex-
pression, 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). Mean-
while,Th17 cells were also not found increased in the mesen-
teric 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 PR8-
specific Th17 cells could not be detected both in the lung and
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.
JEM Vol. 211, No. 12
Ar ticle
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 intes-
tine 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 in-
duce IL-17A expression in both mice and human CD4+ T lym-
phocytes (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 in-
testinal 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 neu-
tralizing 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 im-
mune injury during influenza infection. Further experiments
showed that IL-15 neutralization inhibited IL-17A and IL-6 ex-
pression 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 in-
fluence the changes of the intestinal microbiota (Fig. 8 I), sug-
gesting 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 ex-
ternal invaders. Although much has been learned from studying
2405

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. Unexpect-
edly, 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 non-
mucosal organs such as the liver or kidney. Because this resem-
bles 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 in-
fluenza infection causes intestinal immune injury; furthermore,
these observations provide further evidence to support the ex-
istence 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
Figure 8. Intestinal microbiota induces
Th17 cell polarization in situ via trigger-
ing 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 in-
fection. (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 ex-
pression 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 in-
testine 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 real-
time 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 repre-
sent 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.
particularly at the early stage of infection. Although some stud-
ies suggest that influenza virus disseminates into extrapulmo-
nary 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 tis-
sues 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 in-
fection, 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.
Microbiota cause intestine injury during influenza | Wang et al.

An explosive increase in neutrophils is responsible for
influenza-induced acute lung injury and death. IL-17 is a po-
tent 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 influ-
enza 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 dif-
ferent 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 che-
mokine 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 per-
centage and number of CCR9+CD4+ T cells in both lung and
small intestine increased after influenza infection, and neutral-
izing 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 inflam-
mation of the intestine (Hooper and Macpherson, 2010). How-
ever, 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 fail-
ure 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 po-
larization 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 pro-
posed 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 antibiot-
ics used in neonates increases the risk to develop asthma (Sobko
JEM Vol. 211, No. 12
Ar ticle
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 virus-
specific effector CCR9+CD4+ T cells were recruited into the
small intestine and destroyed the homeostasis of intestinal mi-
crobiota 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 ab-
sence 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 germ-
free and conventional animals. A previous study showed that
commensal SFBs induce IL-6 and IL-23 production to stimu-
late Th17 cell polarization (Ivanov et al., 2009). However, in
our mouse model of respiratory influenza infection, the num-
ber 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 inflamma-
tion occurs in different kinds of mice, we think that the over-
growth 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 inhib-
its 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 guide-
lines at USTC. Mouse-adapted influenza A/PR/8/34 strain (H1N1) was a
gift from H. Meng (Institute of Basic Medicine, Shandong Academy of Med-
ical 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 Petri-
film 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 re-
moved 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 deter-
mine 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 con-
centration 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 (Cloud-
Clone Corp).
Analysis of liver and kidney function. Serum from infected mice or
control mice were collected and stored at 80C until analysis. Liver func-
tion 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-CATCCTGTT-
GTATATGAGGCCCAT-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 per-
formed). 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 manufac-
turers instructions.
Isolation of IEC, IEL, and LPL. IECs were isolated as described in a pre-
vious 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. In-
testines 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 (Invi-
trogen), 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 neutral-
ization, 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 [San-
gon Biotech]) added to their drinking water beginning 4 wk before PR8 in-
fection and continuing until sacrifice, as previously described (Ichinohe et al.,
2011). For intestinal E. coli depletion, mice were treated with 1 mg/ml strep-
tomycin (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 con-
sidered statistically significant when differences achieved values of P < 0.05.
Online supplemental material. Table S1 shows primers used for real-
time 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
Microbiota cause intestine injury during influenza | Wang et al.

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Microbiota cause intestine injury during influenza | Wang et al.
 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
1Institutefor Immunology, Ludwig-Maximilians-University, 80539 Munich, Germany
2Department 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

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 asso-
ciated 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 cyto-
toxic 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
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 homolo-
gous 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
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J. Exp. Med. 2014 Vol. 211 No. 10 2047-2059 2047
www.jem.org/cgi/doi/10.1084/jem.20131333
overexpression of T-bet prevented CD8 T cell exhaustion in
chronic LCMV infection, demonstrating the therapeutic po-
tential of T-bet modulation (Kao et al., 2011). However, the
role of T-bet in human viral infections with dichotomous out-
come 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 differ-
ences between successful versus failing immune response dur-
ing 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 in-
creasingly 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 mech-
anisms 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 re-
solving but deficient in chronic-evolving infection. When
we further characterized the functional correlates behind
these differential expression patterns, we found a strong as-
sociation of T-bet with antigen-specific proliferation and
interferon- production by virus-specific CD8 T cells. In-
duction of T-bet by antigen or IL-2 recovered antigen-
specific 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 suscep-
tible to IL-12 suggests a stepwise mechanism of T cell acti-
vation 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 suc-
cessful CD8 T cell response against HBV and HCV infection
and suggest that impaired induction of T-bet could be an im-
portant mechanism involved in CD8 T cell dysfunction dur-
ing 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 anti-
HBe+ 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 expres-
sion during arHBV was confirmed for 2 additional HBV-
specific 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%; unpub-
lished data). As a proof of principle, we determined antigen-
specific 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 EBV-
specific 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; In-
fluenza A MP 5866) in healthy controls with previously re-
solved 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 major-
ity 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 deter-
mined 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 infec-
tion, frequencies of T-bet+ HCV-specific CD8 T cells were
Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.

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
JEM Vol. 211, No. 10
Ar ticle
Figure 1. HBV- and HCV-specific CD8
T cells express high amounts of T-bet dur-
ing acute spontaneously resolving infec-
tion. PBMCs were isolated from (A) patients
with arHBV, cHBV, rHBV (top), aEBV, pEBV, or
resolved Flu infection (bottom) and (B) pa-
tients with arHCV (top left), rHCV (top right),
acHCV (bottom left), or cHCV infection (bot-
tom right) and analyzed by flow cytometry.
The outlined areas indicate the population of
pentamer+ CD8 T cells and the numbers indi-
cate 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-Whitney-
U 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 percent-
age 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.
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, respec-
tively. 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 pen-
tamer+ 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) accord-
ing to the evolution of viral load between t1 and t3 (Table 1).
No correlation was found between T-bet or Eomes expres-
sion 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 de-
ficiency 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 con-
ditions (Fig. 3, A and D). We found that Eomes+/T-bet
CD8 T cells did not show any significant difference be-
tween arHBV (10%, mean), cHBV (8.9%), arHCV (3%),
Table 1. Kinetics of viral load in acute chronic-evolving HCV
infection
Patient t1 viral load [IU/ml] t3 viral load [IU/ml]
P1 1,300 14
P2 2,980 15
P3 110,000 150
P4 66,000 9,350,000
2050
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 frequen-
cies of T-bet+/PD-1+ virus-specific CD8 T cells were signifi-
cantly 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 virus-
specific 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 fre-
quencies in resolving (means: arHBV, 61.8%; arHCV, 48.9%)
as compared with chronic-evolving infections (cHBV, 5.8%;
Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.
 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.

JEM Vol. 211, No. 10 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 antigen-
specific 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 ad-
dressed the question of whether specific stimulation with anti-
gen (c18-27) or cytokines described as T-bet inducers in
literature (Lighvani et al., 2001; Ylikoski et al., 2005) could in-
crease 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 stimula-
tion 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 consider-
able 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 pa-
tients 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 stimu-
lation 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 (Mann-
Whitney-U test).

2052 Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.


High expression of T-bet is associated
with strong antigen-specific proliferation
Antigen-specific proliferation of CD8 T cells is key to the gen-
eration 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 signifi-
cantly 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 trans-
duction 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
JEM Vol. 211, No. 10
Ar ticle
importance of T-bet in HIV and LCMV clone 13 that univer-
sally establish chronic infection, we assessed its role in human
HBV and HCV infection that allowed us the direct compari-
son 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 in-
fection 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 persis-
tence during long-term chronic infection. As acute HBV in-
fection 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 es-
tablish a definitive association between T-bet expression and
clinical course of HBV infection. All patients who later con-
trolled viral replication had high expression of T-bet in virus-
specific 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 infec-
tions. In the murine LCMV infection model, antigen persis-
tence 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 in-
teraction with viral proteins can occur as well. For example,
HCV core protein inhibits proliferation and interferon- pro-
duction of HCV-specific T cells by blocking their C1q com-
plement 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 chronic-
evolving 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 per-
centage 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- pro- that play an important role in the control of chronic viral infec-
duction in CD8+ T cells of T-bet/ mice and, together with tion (Pearce et al., 2003; Paley et al., 2012). As Eomes might
T-bet, it defines different subsets of virus-specific CD8 T cells compensate for the lack of T-bet in HBV and HCV infection,

2054 Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.


we further analyzed its coexpression with T-bet in virus-specific
CD8 T cells under different conditions. In contrast to the pre-
dominant T-bet+ Eomes population, which was significantly
more frequent in acute resolving versus chronic-evolving in-
fection 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 HBV-
and HCV-specific peripheral blood lymphocytes, whereas the
cited study analyzed HCV-specific intrahepatic lymphocytes
or LCMV-specific lymphocytes in murine infection. Further-
more, a subset of T-bet+ Eomes+ cells was consistently detect-
able 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 ex-
pressed in T-bet+ CD8 T cells supports the notion of a domi-
nant 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 infec-
tion. 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 infec-
tion 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.
JEM Vol. 211, No. 10
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 restim-
ulated 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 stimula-
tion with the respective cytokines. Error bars rep-
resent the SEM. Data are representative of one
experiment due to limited patient material.
**, P < 0.01 (Mann-Whitney-U test).
However, as T-bet was not expressed at later stages of infec-
tion in our analyzed samples we could not assess possible sup-
pressive effects on PD-1 ex vivo.
A successful immune response also depends on the bal-
anced differentiation of CD8 T cells into terminally differen-
tiated 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 ex-
pressed 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 mem-
ory 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 expla-
nation 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 corre-
lation 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 pro-
nounced. Interestingly, CD127 expression on virus-specific
CD8 T cells during acute HCV infection has shown a nega-
tive 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 anti-
gen 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 dif-
ferent 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 inter-
feron- 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 suscepti-
ble to IL-12 (Liao et al., 2011). We sought to clarify the mech-
anism behind the enhanced IFN- induction in IL-12stimulated
CD8 T cells and examined if STAT4, which is known as spe-
cific 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 cyto-
kines, 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 cyto-
kines. This is important, as the synergistic effects of both cy-
tokines 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 treat-
ment of chronic HBV and HCV infection found no signifi-
cant 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 virus-
specific CD4 T cells, CD8 T cells and APC. The various pat-
terns of viral replication seen during acute infection could
2056
result from impairment at different levels of this regulatory net-
work 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 infec-
tion, leading to decreased levels of IL-12, which could contrib-
ute 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 cen-
tral 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 induc-
tion 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.
MATERIALS AND METHODS
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 pa-
tients 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 In-
stitutional Review Board of the medical faculty of the Ludwig-Maximilians-
University (Munich).
Diagnostic criteria. Acute hepatitis was defined as acute onset of nonspe-
cific 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-HBc-
IgM and acute HCV by detection of HCV-RNA or seroconversion of anti-
HCV. 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 re-
mained negative for at least 12 mo. In chronic-evolving acute HCV infec-
tion, 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. Di-
agnosis 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).
Dysfunctional CD8+ T cells in HBV/HCV lack T-bet | Kurktschiev et al.
 Ar ticle

Table 2. Patient characteristics

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

Antibody reagents and viability dyes. The following antibodies were
used for flow cytometry: FITC antiT-bet (clone 4B10), Pacific Blue anti-
CD45RA (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 HPNIE-
EVAL (HLA-B3501). All MHC-I pentamers were PE-labeled and obtained
from ProImmune. All corresponding peptides were synthesized by EMC micro-
collections 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 induc-
tion 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 intracel-
lular markers (T-bet, Eomes, and interferon-). For ex vivo staining, pentamer+
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. Sam-
ples were acquired on a FACSCanto II flow cytometer (BD). Data were ana-
lyzed 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 soft-
ware (GraphPad) and included 2-sided Wilcoxon matched pairs test and
Mann-Whitney-U test for unpaired samples. Statistical significance was de-
fined 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 demon-
strates 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
Accepted: 25 July 2014

JEM Vol. 211, No. 10 2057

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 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
2XiangyaSchool of Medicine, Central South University, Changsha, Hunan Province, 410013, China
3Department 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

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 block-
ade 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, Arm-
strong; cl-13, clone 13; DT,
diphtheria toxin; LCMV, lym-
phocytic choriomeningitis virus;
MFI, mean fluorescence inten-
sity; PD-1, programmed cell
death-1; PD-L1, programmed
cell death ligand-1.
Regulatory T cells expressing transcription fac-
tor Foxp3 are indispensable for preventing im-
mune 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 re-
sponses 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 contrib-
uted equally to this paper.
and facilitate disease progression due to inhibi-
tion 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
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J. Exp. Med. 2014 Vol. 211 No. 9 1905-1918 1905
www.jem.org/cgi/doi/10.1084/jem.20132577
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 posi-
tive 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). Im-
portantly, 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 infec-
tions 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. Be-
sides 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 virus-
specific responses in mice chronically infected with lympho-
cytic 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 modu-
lating CD8 T cell function during persistent antigenic stimu-
lation. In contrast, it has been described that T reg cells are
detrimental to virus-specific T cell responses during persis-
tent 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 character-
ized or fully explored as a therapeutic approach.
To analyze the effects of T reg cells on exhausted virus-
specific CD8 T cells, we used LCMV clone 13 (cl-13) infected
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 spe-
cifically 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 costimula-
tion, 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 res-
cued 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 ac-
companied 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 Arm-
strong (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)

1906 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 recogniz-
ing 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 sev-
eral cell surface molecules. CD25 expression was not signifi-
cantly 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 in-
fected mice appeared more activated (Fig. 1 E).
To examine the role of T reg cells during chronic infec-
tion, we infected Foxp3DTR knock-in mice with LCMV cl-13
after antibody-mediated transient depletion of CD4 T cells,
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 re-
sults show that during chronic infection, T reg cell depletion
enables expansion of previously exhausted T cells.
In addition to expansion, the surface phenotype of ex-
hausted 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

JEM Vol. 211, No. 9 1907

 Figure 3. LCMV-specific CD8 T cells re-
gain effector function upon T reg cell ab-
lation 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 num-
bers 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 DbGP276-
specific 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 DbG276-
specific CD8 T cells in spleen. (G) Ex vivo cyto-
toxic activity of splenic CD8 T cells measured
by 51Cr release from MC57 target cells un-
pulsed (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 induces CD127 expression on exhausted CD8 T cells during
promote their long-term survival but is down-regulated on chronic LCMV infection (West et al., 2013). Hence, it is pos-
exhausted T cells (Kaech et al., 2003; Wherry and Ahmed, sible that an increase in IL-2 availability upon T reg cell abla-
2004). Interestingly, a recent report described that IL-2 therapy tion 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 chron-
ically infected Foxp3DTR knock-in mice were de-
pleted 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 treat-
ment. (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 indepen-
dent 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 T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.
 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 ab-
solute numbers of virus-specific CD8 T cells that could pro-
duce IFN- upon stimulation with LCMV peptides (Fig. 3 A).
T reg cell depletion not only affected the number of LCMV-
specific 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 LCMV-
specific 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 se-
verely 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 res-
cued 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.
For example, blockade of the PD-1 inhibitory pathway in-
creased 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). Simi-
lar to mice chronically infected with LCMV, T reg cell abla-
tion triggered expansion of total activated CD4 and CD8

JEM Vol. 211, No. 9 1909


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 LCMV-
specific 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
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 abla-
tion. (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 experi-
ments with 35 mice per group. Error bars indicate
SEM. Non-parametric Mann Whitney test, where
*, P < 0.05; **, P < 0.01; ***, P < 0.001.
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
T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.
 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 path-
way. 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 pre-
vented the increase in the response of virus-specific CD8
T cells that ensues upon T reg cell ablation in chronically in-
fected mice. These results imply a critical role for costimula-
tion 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 deple-
tion (Kim et al., 2007). To address the role of conventional
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 pres-
ents antigens to CD4 T cells (Dudziak et al., 2007). Impor-
tantly, CD4 T cell depletion completely abrogated the rescue
of LCMV-specific CD8 T cells observed upon T reg cell abla-
tion 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 se-
lection (Matloubian et al., 1994). Therefore, it is highly un-
likely 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.

JEM Vol. 211, No. 9 1911


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 LCMV-
specific CD8 T cells was associated with viral control. Unex-
pectedly, we did not observe a significant reduction in viral
load in chronically infected mice (Fig. 7 A) despite the im-
pressive rescue of LCMV-specific CD8 T cell responses in-
duced by T reg cell depletion. Antiviral T cells are known to
control virus by direct killing of infected cells and by produc-
tion 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
Figure 8. Essential role for PD-L1 block-
ade in viral control after T reg cell deple-
tion. 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) Abso-
lute 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 com-
pilation (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.
(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 in-
flammatory cytokine levels in the serum of LCMV chroni-
cally 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 compari-
son with their uninfected counterparts (Fig. 7 E). In addition
to DCs, LCMV cl-13 replicates extensively in nonhemato-
poietic cells, such as fibroblastic reticular cells, endothelial
cells, and diverse parenchymal cell types, and PD-L1 expres-
sion in these cells is known to restrict viral control (Mueller
T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.

et al., 2007, 2010; Frebel et al., 2012). Thus, we reasoned that
PD-1 interactions with the increased levels of PD-L1 ex-
pressed by infected cells might oppose viral clearance by func-
tionally 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 de-
pletion. 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 com-
bination with the PD-1 pathway blockade was similar (Fig. 8,
CE), with the exception of an additive increase in the fre-
quency 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
Ar ticle
Figure 9. Transient T reg cell depletion
improves CD8 T cell rescue and viral con-
trol 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 treat-
ment, relative to initial weight before treat-
ment. (B) As in A, but mice received transient
DT treatment (on days 0, 1, and 4). Mice
weight after 12 d of treatment. (C) Represen-
tative dot plots show frequency of T reg cells
in PBMC before and after continuous or tran-
sient 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.
rescue. Because T reg cell depletion resulted in a greater num-
ber of functional virus-specific T cells when compared with
PD-1 blockade alone (Fig. 8, CE), and the combined ther-
apy further improved viral control (Fig. 8, A and B), our data
support the use of combination therapies based on modula-
tion 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 de-
crease in viral load are not always directly correlated. These
results show that target cell elimination is affected both by in-
trinsic 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 administra-
tion, 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 re-
sulted 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 sin-
gle 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 LCMV-
specific 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 combina-
tion 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 res-
cued 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 combi-
nation 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 re-
duce viral burden. When we tried to understand this unex-
pected 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 combina-
tion 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 chroni-
cally 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 proin-
flammatory 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 func-
tion (Wing et al., 2008). It has been shown that CTLA-4 in-
teracts 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 stimula-
tion 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 ex-
hausted CD8 T cells (Barber et al., 2006), suggesting that
CTLA-4 does not constitute a major nonredundant suppres-
sive 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 gener-
alized 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 deple-
tion of conventional CD4 T cells abrogated activation of splenic
CD11b+ DCs triggered by T reg cell ablation in LCMV chron-
ically 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 abla-
tion. There might be important differences between DC sub-
sets 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
T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.

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 identifica-
tion 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 mem-
ory 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 deple-
tion and blockade of the PD-1 pathway on the antiviral re-
sponse 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 abla-
tion, 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. Be-
sides 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). Interest-
ingly, we could not detect any significant amount of inflam-
matory 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
Ar ticle
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 molecu-
lar mechanisms involved in rescue of T cell exhaustion.
Importantly, our study shows that even a 10100-fold in-
crease 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 virus-
infected 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 improve-
ment 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 antibod-
ies, 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 de-
pletion 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 fur-
ther increased by several cytokines, including IFN- (Dong
et al., 2002; Keir et al., 2008).We showed that T reg cell deple-
tion 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, es-
pecially 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 detri-
mental effect on the course of the infection (Teijaro et al.,
2013; Wilson et al., 2013). Thus, therapies that promote cyto-
toxic T cell responses still need to ensure that further inhibi-
tory mechanisms will not hamper destruction of target cells.
Reversal of T cell exhaustion is the ultimate goal of im-
munotherapies 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 im-
prove synergy to achieve better response rates as well as lon-
gevity 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 monother-
apy trials. Thus, our results provide important concepts that
may have direct relevance to cancer treatment. Nevertheless, ad-
ditional studies need to be done to determine which chronic
infections and cancers would be responsive to this combina-
torial immunotherapy involving T reg cell depletion and
blockade of the PD-1 pathway because there may be hetero-
geneity 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 con-
trol.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 VEGF-
producing tumors (Hansen et al., 2012), have also been pro-
posed 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 pre-
sented here also underscore a critical role of PD-L1 in deter-
mining viral control after T cell rescue from an exhausted state.
MATERIALS AND METHODS
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 infec-
tions 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 infec-
tion. Titration of virus was performed on Vero cell monolayers as described
previously (Ahmed et al., 1984). All mouse experiments were performed ac-
cording to institutional guidelines and were approved by the Emory Univer-
sity 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 fi-
broblast 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 = ef-
fector 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 calcu-
late 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 sus-
pensions 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 tetra-
mer (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. Intracel-
lular staining for IFN-, TNF, Ki67, and granzyme B were performed with
the Cytofix/Cytoperm kit (BD). Intracellular staining of Foxp3 was per-
formed according to manufacturers instructions (eBioscience). Serum cyto-
kines 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.
T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.

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
Accepted: 10 July 2014
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T reg cells and PD-1 modulate T cell exhaustion | Penaloza-MacMaster et al.
INSIGHTS

CCR4 dr ives ATLL jail break

Adult T cell leukemia/lymphoma (ATLL), caused by human T cell lymphotropic virus 1 (HTLV-1), is
an aggressive cancer that is refractory to current therapies. The long latency and low overall penetrance of
ATLL in HTLV-1infected individuals infers the need for cooperating events, which include somatic
JAK3, NOTCH1, and FAS mutations. While overexpression of CCR4 is a hallmark of ATLL, it is not clear
whether dysregulation of CCR4 function contributes to disease pathogenesis. In this issue, Nakagawa et
al. report recurrent somatic mutations in the CCR4 chemokine receptor in ~25% of ATLL cases, impli-
cating these mutations in ATLL pathogenesis.
The authors performed RNA transcriptome analysis of two ATLL cases and targeted sequencing of addi- Insight from
tional ATLL patient samples and cell lines. Remarkably, the CCR4 mutations found in primary ATLL speci- Kevin Shannon
mens were heterozygous and introduced missense or truncating mutations in a conserved carboxy-terminal
domain of CCR4 involved in negative regulation. Together, these findings suggested a dominant gain-of-function mechansim of
action. Indeed, elegant functional studies revealed defective internalization of these mutant CCR4 proteins as well as enhanced
migration and chemotaxis in response to chemokines. The authors also demonstrated hyperactive PI3 kinase/Akt signaling in
ATLL cells expressing CCR4 mutant proteins.
So, how do CCR4 mutations promote malignant growth? The authors provide two logical and nonexclusive explana-
tions. First, these mutations might enable ATLL cells to migrate to and colonize niches in tissues such as skin and lymph nodes
that are favorable for cancer cell survival and proliferation. If this
idea is correct, it is possible that patients with and without CCR4
mutations will exhibit specific patterns of tissue involvement and
disease evolution. Second, dysregulated PI3K signaling down-
stream of mutant CCR4 might be the key biochemical driver con-
tributing to clonal selection of ATLL cells. Given this underlying
biology, it is reasonable to speculate that seed and soil both
contribute to the aberrant growth of ATLL tumors with somatic
CCR4 mutations. For example, because CCR4 mutations render
PI3K signaling hypersensitive to chemokine stimulation, specific
tissue microenvironments likely favor ATLL growth through para-
crine mechanisms.
An exciting aspect of these new mechanistic insights is their
potential for clinical translation. A first generation anti-CCR4
antibody called KW-0761 is showing promise in early phase clinical
trials. It will be interesting to determine whether mutant CCR4 is
Schematic of CCR4 mutant isoforms in ATLL a predictive biomarker of sensitivity to this and other anti-CCR4
agents. Deep genomic analysis of tumors with CCR4 mutations
that relapse after an initial response will provide additional insights. PI3 kinase inhibitorsboth alone and in combination with
other agentsare another potential therapeutic strategy for improving outcomes in this relentless cancer.
Nakagawa, M., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140987.

Kevin M. Shannon, University of California, San Francisco: shannonk@peds.ucsf.edu


 Brief Definitive Report

Gain-of-function CCR4 mutations in adult

T cell leukemia/lymphoma
Masao Nakagawa,* Roland Schmitz,* Wenming Xiao, Carolyn K. Goldman,
Weihong Xu, Yandan Yang, Xin Yu, Thomas A. Waldmann,**
and Louis M. Staudt**

Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Bethesda, MD 20892

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell

lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the
pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL
patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemo-
kine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and con-
sisted exclusively of nonsense or frameshift mutations that truncated the coding region at
C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense
isoform was gain-of-function because it increased cell migration toward the CCR4 ligands
CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced
PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and con-
ferred a growth advantage in long-term in vitro cultures. These findings implicate somatic
gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition
of CCR4 signaling might have therapeutic potential in this refractory malignancy.

CORRESPONDENCE
Louis M. Staudt:
lstaudt@mail.nih.gov
Abbreviations used: ATLL,
adult T cell leukemia/
lymphoma; CCR4, CC
chemokine receptor 4; GPCR,
G proteincoupled receptor;
huKO, human Kusabira-
Orange; PI3K, PI(3) kinase;
PTX, pertussis toxin; SNV,
single nucleotide variant.
Adult T cell leukemia/lymphoma (ATLL) is
one of the most aggressive forms of peripheral
T cell lymphoma, with a median survival of
<1 yr with current therapy, which consists pri-
marily of cytotoxic chemotherapy (Campo et al.,
2011). Molecular analyses of ATLL cells re-
vealed that high expression of CC chemokine
receptor 4 (CCR4) is a hallmark of this disease
(Yoshie et al., 2002; Ishida et al., 2003; Iqbal
et al., 2010). Clinical trials in ATLL of a thera-
peutic monoclonal antibody directed against
CCR4 (KW-0761) are ongoing, and promising
early results have been reported (Yamamoto et al.,
2010; Ishida et al., 2012).
CCR4 is a chemokine receptor that has a
critical role in immune cell trafficking.T-helper
type 2 cells (Th2), regulatory T cells (Treg),
interluekin-17producing T-helper cells (Th17),
and skin-homing memory T cells express CCR4
on their surface and migrate toward the che-
mokines CCL17 and CCL22 (Imai et al., 1997,
1998; Yoshie, 2005). The leukemic cells in 90%
of ATLL cases express CCR4 on their surface
(Ishida et al., 2003). Interestingly, the most
frequent sites of ATLL involvement are lymph
nodes and skin (Campo et al., 2011), where den-
dritic cells, M2-phenotype macrophages, Lang-
erhans cells, and cutaneous venules can produce
CCL17 and/or CCL22 (Campbell et al., 1999;
Vissers et al., 2001; Vulcano et al., 2001; Chong
et al., 2004). These observations suggest that
CCR4 could have a role in ATLL biology, but it
is still unclear whether dysregulation of CCR4
function contributes to ATLL pathogenesis.
Human T cell lymphotropic virus type-I
(HTLV-I) is believed to be the causative agent
for ATLL (Matsuoka and Jeang, 2007; Campo
et al., 2011). However, only a small proportion
of HTLV-I carriers (27%) develop ATLL with
a long latency (4050 yr; Arisawa et al., 2000;
Campo et al., 2011). Thus, acquisition of so-
matic mutations in cellular genes is likely to be
crucial for the development of ATLL. Identi-
fying such somatic mutations is essential not
only for understanding ATLL pathogenesis but
also for defining molecular targets for therapy.

This article is distributed under the terms of an AttributionNoncommercial

*M. Nakagawa and R. Schmitz contributed equally to this paper. Share AlikeNo Mirror Sites license for the first six months after the publication
date (see http://www.rupress.org/terms). After six months it is available under a
**T.A. Waldmann and L.M. Staudt contributed equally to Creative Commons License (AttributionNoncommercialShare Alike 3.0 Unported
this paper. license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

The Rockefeller University Press $30.00

J. Exp. Med. 2014 Vol. 211 No. 13 24972505 2497
www.jem.org/cgi/doi/10.1084/jem.20140987
Somatic mutations in p53, NOTCH1, JAK3, and FAS have
been reported in ATLL (Elliott et al., 2011; Yamagishi and
Watanabe, 2012), but our knowledge of genetic aberrations in
this malignancy is nonetheless incomplete.
In the present study, we used whole transcriptome analy-
sis (RNA-seq) to discover activating mutations in CCR4,
which we found to be a frequent genetic event in this malig-
nancy. Functional analysis clarified the gain-of function nature
of these mutations, suggesting that dysregulation of CCR4
function is key to the pathogenesis of ATLL.
RESULTS AND DISCUSSION
Frequent mutation of CCR4 in ATLL
We performed RNA-seq of peripheral blood leukemia sam-
ples from two ATLL patients, TW36R and TW51R, which
allowed us to identify 85 and 127 genes with potential coding
region mutations in these two samples, respectively. These
candidates included two genes that were mutated in both
samples: CCR4 and MICALL1. High expression of CCR4 is
a well-known hallmark of ATLL, whereas MICALL1 has not
been implicated in this disease. Importantly, both ATLL sam-
ples had the same nonsense CCR4 mutation affecting the
Y331 codon (Y331*), suggesting that CCR4 mutations might
play a critical role in ATLL pathogenesis. The percentage of
mutant CCR4 sequencing reads was 39% in TW36R and
55% in TW51R. Because both blood samples had a high pro-
portion of malignant cells (TW36R: 90%, TW51R: 84%), the
CCR4 mutations are likely to be heterozygous and poten-
tially might exert a dominant functional effect.
By Sanger sequencing of genomic DNA, we confirmed
the heterozygous nature of the CCR4 nonsense mutations in
TW36R and TW51R. We extended this analysis to an addi-
tional cohort of ATLL primary patient samples (n = 41)
and ATLL cell lines (n = 12). CCR4 mutations were de-
tected in 26.4% (14/53) of ATLL samples (Fig. 1, A and B;
and Table S1). In five cases for which paired normal DNA
was available, three different CCR4 mutations were detected
only in the ATLL cells (Q330*, Q330 frameshift, and Y331*),
demonstrating that they were acquired somatically during
malignant transformation or progression (Fig. 1 C). CCR4
mutations were identified in both primary ATLL patient samples
(24%, 10/41) and in ATLL cell lines (33.3%, 4/12). Muta-
tions were heterozygous in all samples except one cell line
(ATL42T+). We also confirmed that mutant CCR4 mRNAs
were heterozygously expressed in six primary ATLL sam-
ples (Table S1). These findings indicated that CCR4 muta-
tions in ATLL might be either gain-of-function or potentially
dominant negative.
All CCR4 mutations were either nonsense or frameshift
in nature and affected the codons for four nearby amino acids
(F326, C329, Q330, and Y331) that are located in an evolu-
tionarily conserved region in the carboxy terminus of the
protein (Fig. 1, A and B). The most frequent nonsense muta-
tion affected codon Y331 (Y331*; 7/53, 13%). Four cell lines
(ED40515(+), ED40515(), ED41214(+), and ED41214 C())
had Q330* nonsense mutations, which is understandable given
2498
that these four lines were established from the same patient
at different clinical time points.
In cancer genome studies cataloged in the COSMIC data
base (Catalogue of somatic mutations in cancer) or con-
ducted by The Cancer Genome Atlas (TCGA) initiative, we
found only three primary human cancer samples with CCR4
nonsense mutations, and these mutations did not target the
carboxy-terminal cytoplasmic domain. Thus, truncation of
the CCR4 cytoplasmic domain by somatic mutation appears
to be a specific and frequent genetic event to ATLL.
Mutant CCR4 enhances chemotaxis toward CCR4 ligands
Chemokine receptors, including CCR4, belong to the seven-
transmembrane G proteincoupled receptor (GPCR) family.
All CCR4 mutations in ATLL encode truncated receptors that
lack most of the carboxy-terminal cytoplasmic domain, which
typically serves a regulatory role in GPCRs (Fig. 1, A and B;
Luttrell and Lefkowitz, 2002). The loss of this region of CCR4
might deregulate its activity, potentially altering the migration
of ATLL cells in response to CCR4 ligands. The CCR4 muta-
tions in ATLL are reminiscent of mutations affecting the
chemokine receptor CXCR4 in WHIM syndrome, a human
immunodeficiency disease (Diaz, 2005). Most CXCR4 muta-
tions in WHIM syndrome are nonsense or frameshift mu-
tations that truncate the carboxy-terminal cytoplasmic domain
of the protein, conferring a gain-of-function phenotype
with respect to chemotaxis toward the CXCR4 ligand SDF1
(Balabanian et al., 2005; Diaz, 2005; Kawai et al., 2005).
We therefore hypothesized that mutant CCR4 isoforms
might enhance chemotaxis of the affected cells to CCR4 li-
gands. To study this, we infected a mouse myeloid cell line,
32D, with retroviral vectors expressing WT CCR4 (CCR4-
WT) or CCR4-Q330* coding regions and tested the migra-
tion of the transduced cells toward CCL22, the most potent
CCR4 ligand (Fig. 2 A; DAmbrosio et al., 2002). Surface CCR4
levels were similar between CCR4-WT and CCR4-Q330*
transduced 32D cells, whereas mock vectortransduced 32D
cells did not have detectable CCR4 expression (Fig. 2 B). Mock
vectorinfected 32D cells did not migrate toward CCL22
(Fig. 2, A and B). CCR4-WTtransduced 32D cells mi-
grated with a typical bell-shaped doseresponse curve (Fig. 2,
A and B). Compared with CCR4-WT, CCR4-Q330*
transduced 32D cells migrated to a significantly greater ex-
tent (Fig. 2, A and B). These results support the view that the
CCR4 mutants in ATLL are gain-of-function with respect to
ligand-directed chemotaxis.
Next we evaluated the ability of CCR4 isoforms to me-
diate chemotaxis in ATLL cell lines (Fig. 2 C). Most ATLL
cell lines express CCR4 on their surface (not depicted), re-
flecting the high frequency (90%) of CCR4 expression in
primary ATLL cases (Ishida et al., 2003). To evaluate CCR4-
WT and CCR4-Q330* in ATLL cells, we first knocked
down the endogenous expression of CCR4 in ED40515(+)
cells using an shRNA targeting the 3 untranslated region
(UTR) of the CCR4 mRNA. This resulted in a >90% reduc-
tion of surface CCR4 expression (Fig. 2 D) and substantially
Gain-of-function CCR4 mutations in ATLL | Nakagawa et al.

Figure 1. CCR4 mutations in ATLL. (A) Amino acid residues in the carboxy-terminal region of CCR4. (B) Schematic of CCR4 mutant isoforms in ATLL.
(C) DNA sequence of the CCR4-Y331* mutation in the TW14R ATLL biopsy sample by Sanger sequencing (bottom). The WT sequence of normal DNA obtained
from the same patient is shown in the top.
decreased chemotaxis of the cells (Fig. 2 C). Cells were then
transduced with retroviruses expressing CCR4-WT or
CCR4-Q330* coding regions, resulting in cell populations
with equivalent expression of CCR4 on the cell surface
(Fig. 2 D). In chemotaxis assays with CCL17, CCR4-WT
reconstituted cells exhibited greater dose-dependent migra-
tion than mock vectorreconstituted cells, but migration of
CCR4-Q330*reconstituted cells was significantly greater
than either of the other populations (Fig. 2 C, left). In re-
sponse to CCL22, the most potent CCR4 ligand (DAmbrosio
et al., 2002), CCR4-Q330*reconstituted ED40515(+) cells
again displayed significantly greater chemotaxis than CCR4-
WTreconstituted cells and responded better to lower con-
centrations of the ligand (Fig. 2 C, right). Because CCR4
mutations in ATLL samples were heterozygous, we also
analyzed whether these mutations could enhance chemotaxis
in the presence of WT CCR4. To this end, we transduced
the shCCR4-ED40515(+) line with one retroviral vector
that coexpresses CCR4-WT and the human Kusabira-Orange
(huKO) fluorescent protein and another vector that co
expresses CCR4-Q330 and GFP. This strategy allowed us to
compare the phenotype of GFP/huKO double-positive cells
that expressed CCR4-WT and Q330* with GFP or huKO
single-positive cells that only ectopically expressed one CCR4
isoform (Fig. 2, E and F). Cells expressing both CCR4-WT
and CCR4-Q330* showed greater CCL22-directed mi-
gration than cells expressing CCR4-WT alone, which was
similar to the phenotype of cells expressing CCR4-Q330*
alone (Fig. 2 F). These results suggest that ATLL cells may
acquire CCR4 mutations to migrate more effectively toward
their ligands.
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Br ief Definitive Repor t
Mutant CCR4 impairs receptor internalization
after CCL22 binding
The carboxy-terminal region of CCR4 that is truncated by
mutations in ATLL contains a serine- and threonine-rich
motif that is shared by many GPCRs (Fig. 1 A, amino acid
positions 342351). These serine and threonine residues
become rapidly phosphorylated in response to ligand, which
results in receptor internalization and contributes to desen-
sitization of the cells to ligand (Luttrell and Lefkowitz,
2002). Carboxy-terminal truncation of CXCR4 in WHIM
syndrome impairs receptor internalization, contributing to
the enhanced migration of these cells in response to ligand
(Balabanian et al., 2005; Kawai et al., 2005).
We therefore studied the change in surface CCR4 levels
after CCL22 exposure in CCR4-WT and CCR4-Q330*
reconstituted ED40515(+) cells. In CCR4-WTreconstituted
cells, surface CCR4 levels declined rapidly, with a 58% re-
duction at 5 min after CCL22 exposure and a maximum re-
duction of 75% at 20 min (Fig. 2 G). In comparison, CCR4
internalization in CCR4-Q330*reconstituted cells was
significantly impaired, with a 27% of reduction at 5 min and
reaching only a 54% reduction at 20 min (Fig. 2 G). These
results suggest that the ATLL CCR4 mutants impair desensi-
tization by ligand, which likely contributes to the enhanced
chemotaxis of cells bearing these mutants.
Mutant CCR4 enhances PI(3) kinase (PI3K)/AKT signaling
in response to ligand
We explored the influence of the ATLL CCR4 mutants on
PI3Kdependent activation of AKT because it has been
2499
Figure 2. CCR4 mutant isoforms enhance chemotaxis and impair receptor internalization. (A) CCL22-mediated chemotaxis of mouse 32D cells
ectopically expressing CCR4-WT or CCR4-Q330*. In these Transwell assays, the lower chamber contained the indicated amount of CCL22. After a 2-h incuba-
tion, the number of cells migrating from the upper to lower chamber was determined and plotted as a percentage of the input cell number. (B) Surface CCR4
expression levels of 32D cells ectopically expressing CCR4-WT or CCR4-Q330* analyzed by FACS. (C) Chemotactic ability of CCR4-WT or CCR4-Q330*
reconstituted ED40515(+) ATLL cells toward CCL17 and CCL22. (D) Surface CCR4 expression levels in CCR4-WT or CCR4-Q330*reconstituted ED40515(+)
ATLL cells analyzed by FACS. (E) Surface CCR4 expression levels analyzed by FACS in ED40515(+) ATLL cells ectopically expressing CCR4-WT and/or CCR4-Q330*.
(B, D, and E) Isotype control IgG staining is indicated in gray. (F) CCL22-induced chemotaxis of ED40515(+) ATLL cells ectopically expressing CCR4-WT and/or
CCR4-Q330*. (G) Time course of surface CCR4 levels after CCL22 exposure in CCR4-WT or CCR4-Q330*reconstituted ED40515(+) ATLL cells. Surface CCR4
levels were analyzed by FACS and normalized to the levels at time 0. Data in all panels are presented as mean SEM of technical duplicates representative of
at least two biological replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for a comparison between CCR4-WT and CCR4-Q330*.

reported that binding of CCL22 to CCR4 activates AKT in
CEM leukemic T cells and in human Th2 cells (Cronshaw
et al., 2004). We first studied two ATLL cell lines: ED40515(+),
which bears a CCR4-Q330* mutant allele, and KOB, in
which CCR4 is WT. Immunoblot analysis revealed that
baseline levels of phospho-AKT (P-AKT), a measure of AKT
activation, were much lower in ED40515(+) than in KOB
(Fig. 3 A, lane 2 vs. lane 6). However, ED40515(+) showed
stronger induction of AKT phosphorylation at 10 min after
CCL22 exposure than KOB (Fig. 3 A, lane 3 vs. lane 7). The
activation of AKT was transient in both cell lines, decreasing
by 30 min after stimulation (Fig. 3 A, lanes 4 and 8). These
findings indicated that AKT activation is one of the signal-
ing pathways downstream of CCR4 in ATLL cells and sug-
gested that CCR4 mutations might affect the magnitude
of AKT activation. To accurately evaluate the relative ability
of CCR4 isoforms to activate AKT, we studied ED40515(+)
cells that were transduced with a CCR4 shRNA to knock
down endogenous CCR4 expression and were reconstituted
with CCR4-WT or CCR4-Q330*. Cells were treated with
CCL22, and P-AKT levels were measured by FACS at vari-
ous time points after exposure (Fig. 3 B). Knockdown of
endogenous CCR4 reduced AKT activation by CCL22 com-
pared with mock-infected cells, confirming that AKT activation
was dependent on CCR4. Ectopic provision of CCR4-WT
did not restore AKT activation despite expression of CCR4
at a higher level than in mock-transduced ED40515(+)
cells (Fig. 2 D), presumably because the endogenous CCR4
locus in ED40515(+) encodes the CCR4-Q330* isoform.
In contrast, cells reconstituted with CCR4-Q330* restored
robust AKT activation in response to CCL22 (Fig. 3 B). In
KOB cells, reconstitution with CCR4-WT modestly in-
creased P-AKT activation in response to CCL22, but cells
reconstituted with CCR4-Q330* again responded with a
significantly greater rise in P-AKT levels (Fig. 3, C and D).
The effect of heterozygous mutation of CCR4 on AKT
activation was analyzed using the dual fluorescence strat-
egy described above for experiments depicted in Fig. 2
(E and F). After CCL22 treatment, ED40515(+) cells ex-
pressing both CCR4-WT and CCR4-Q330* or CCR4-
Q330* alone had elevated p-AKT levels compared with
cells expressing only CCR4-WT (Fig. 3 E). Together, these
data demonstrate that CCR4 mutations in ATLL enhance
AKT activation in response to ligand engagement.

2500 Gain-of-function CCR4 mutations in ATLL | Nakagawa et al.


We next investigated whether AKT activation is involved
in CCR4-mediated chemotaxis in ATLL. The pan-PI3K in-
hibitor BKM120 abrogated CCR4-mediated AKT activation
in ED40515(+) cells in a dose-dependent manner, indicating
that PI3K signaling contributes to AKT activation (Fig. 3 F).
However, BKM120 treatment only partially inhibited CCR4-
mediated chemotaxis by ED40515(+) cells (Fig. 3 G). In con
trast, the Gi inhibitor pertussis toxin (PTX) abrogated AKT
activation and chemotaxis in these cells (Fig. 3, F and G).
Thus, CCR4-mediated chemotaxis of ATLL cells is not pri-
marily caused by PI3K-dependent AKT signaling, but instead
depends on Gi, consistent with previous work (Cronshaw
et al., 2004).
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Br ief Definitive Repor t
Figure 3. Enhanced PI3K/AKT activation by mutated
CCR4. (A) Immunoblot analysis for P-AKT levels in
ED40515(+) and KOB ATLL cells after CCL22 exposure
(50 ng/ml). Blots using P-AKT (S473) antibody are shown
with short and long exposure times. Relative scanning den-
sitometry estimates of P-AKT levels are depicted under the
panel showing the short exposure. The pan-PI3K inhibitor
BKM120 was used at 1 M. (B and C) Time course experi-
ment of AKT activation after CCL22 exposure (50 ng/ml)
using CCR4-reconstituted ED40515(+) cells (B) and KOB
cells (C), transduced as indicated with shRNA and cDNA
expression vectors. P-AKT (S473) levels were analyzed by
FACS at the indicated times after CCL22 exposure. MFI,
mean fluorescence intensity. (D) Surface CCR4 expression
levels in CCR4-WT or CCR4-Q330*reconstituted KOB cells.
Isotype control IgG staining is indicated in gray. (E) Time
course experiment of AKT activation after CCL22 expo-
sure (50 ng/ml) in ED40515(+) ATLL cells ectopically ex-
pressing CCR4-WT and/or CCR4-Q330*. (F) P-AKT (S473)
levels of ED40515(+) treated with inhibitors. Cells were
pretreated with 100 ng/ml PTX for 16 h or the indicated
amount of the pan-PI3K inhibitor BKM120 for 1 h and then
incubated with 50 ng/ml CCL22 for 5 min. P-AKT (S473) was
analyzed by FACS. (G) Chemotaxis of ED40515(+) cells
treated with inhibitors. Cells were pretreated with PTX or
BKM120 as in F and then used in a CCL22-mediated chemo-
taxis assay. Data in all panels are presented as mean SEM
of technical duplicates representative of at least two bio-
logical replicates. *, P < 0.05; **, P < 0.01 for a comparison
between CCR4-WT and CCR4-Q330*.
Mutant CCR4 promotes ATLL expansion
in the presence of ligand
Lastly, we tested whether the acquisition of CCR4 mutations
by ATLL cells imparts a selective growth advantage relative
to cells with WT CCR4. After shRNA-mediated knockdown
of endogenous CCR4 expression, ED40515(+) cells were
transduced with a retrovirus expressing CCR4-WT together
with GFP or with a retrovirus expressing only CCR4-Q330*
(Fig. 4 A, Exp. 1). After puromycin selection of infected cells,
these two cell populations were mixed in equal numbers and
cultured with or without CCL22 for 12 d. The ratio of GFP/
CCR4-Q330*expressing cells versus GFP+/CCR4-WT
expressing cells was monitored every 4 d by FACS (Fig. 4 B,
2501

Exp. 1). Without CCL22, the ratio of the two populations
did not change. In contrast, in the presence of CCL22, CCR4-
Q330*reconstituted cells preferentially expanded in a time-
dependent manner. To confirm this finding, we performed
a GFP-swapping experiment in which the GFP+ cells ex-
pressed CCR4-Q330* and the GFP cells expressed CCR4-
WT (Fig. 4 A, Exp. 2). Again, CCR4-Q330*reconstituted
cells had a selective growth advantage in the presence of
CCL22 (Fig. 4 B, Exp. 2). As a negative control, we used
empty vectors to create GFP+ and GFP populations and
observed no change in the GFP+/GFP ratio in the presence
or absence of CCL22 (Fig. 4, A and B, Exp. 3).
In conclusion, the present study demonstrates that muta-
tions in CCR4 are a frequent genetic event in ATLL and pro-
vides a mechanistic rationale for their selection in this cancer.
Mutant CCR4 isoforms enhanced migration of ATLL cells
toward CCL17 and CCL22 and additionally promoted PI3K/
AKT activation in response to ligand engagement, leading us
to propose two nonmutually exclusive hypotheses regarding
the role of CCR4 mutations in ATLL pathogenesis. First,
enhanced migration of ATLL cells along a CCL17/CCL22
gradient might allow them to access a favorable microenvi-
ronmental niche that could support their proliferation and/or
survival. Lymph nodes and skin are plausible ATLL niches
because CCL17 and CCL22 are known to be produced by
dendritic cells and M2-phenotype macrophages in lymph nodes
and also by Langerhans cells and venules in skin (Campbell et al.,
2502
Figure 4. Growth advantage of CCR4-Q330*
reconstituted ATLL cells. (A) Schematic of the competitive
growth assay. ED40515(+) cells depleted of endogenous
CCR4 expression by RNA interference were transduced with
the indicated CCR4-expressing vectors or mock vectors. After
puromycin selection, two transduced populations, one ex-
pressed GFP and the other did not, were mixed equally and
co-cultured for 12 d in vitro. The ratio of the two popula-
tions was determined by FACS every 4 d. (B) CCR4-Q330*
transduced cells have a competitive growth advantage.
CCL22 was added every 2 d at 50 ng/ml. The ratios of the
two populations were normalized to the value at day 0.
Growth curves represent the mean of eight replicates ob-
tained from four biologically independent experiments SEM.
*, P < 0.05; ***, P < 0.001.
1999; Vissers et al., 2001; Vulcano et al., 2001; Chong et al.,
2004). Previous studies showed that monocyte and M2-
phenotype macrophages can promote ATLL cell growth through
cellcell interaction and by paracrine mechanisms (Chen
et al., 2010; Komohara et al., 2013). Thus, the chemotaxis
promoted by CCR4 mutations in ATLL might set up favor-
able interactions between ATLL cells and immune bystander
cells and/or disrupt homeostatic mechanisms that keep the
growth of these cells in check. Recently, WHIM syndrome-
like CXCR4 somatic mutations have been detected in the
malignant cells of patients with Waldenstrms macroglob-
ulinemia, and these mutations correlated with bone mar-
row involvement as well as chemotherapy drug resistance
(Roccaro et al., 2014; Treon et al., 2014). Given that CCR4
mutations and WHIM syndromelike CXCR4 mutations
share similar biological properties in terms of chemotaxis and
ATK activation (Cao et al., 2014; Treon et al., 2014), it will
be interesting to determine whether CCR4 mutations affect
the frequency of lymph node or skin involvement in ATLL or
the clinical course.
A second hypothesis raised by our findings is that CCR4
mutations may be selected in ATLL for their ability to
enhance signaling downstream of this chemokine receptor,
including activation of the PI3K/AKT signaling pathway.
During physiological CCR4 signaling, ligand desensitization
occurs in part because CCR4 is down-modulated from the cell
surface, a process which is disrupted by the CCR4 mutations
Gain-of-function CCR4 mutations in ATLL | Nakagawa et al.

in ATLL. Perhaps as a result, ATLL cells bearing CCR4 mu-
tant isoforms displayed prolonged PI3K/AKT activation in
response to ligand. Given the importance of PI3K/AKT sig-
naling to both cellular metabolism and survival, this enhanced
PI3K/AKT response might provide a selective advantage for
ATLL cells. Indeed, in our long-term competitive growth
assay, ATLL cells expressing mutant CCR4 outgrew cells
with WT CCR4 in the presence of CCR4 ligand. Finally, it
is conceivable that both hypotheses detailed above may per-
tain. Specifically, the ability of CCR4 mutants to increase
chemotaxis toward CCR4 ligands would expose them to higher
ligand concentrations, which might contribute to their growth
and/or survival.
Our findings provide a rationale to test whether inhibi-
tion of CCR4 signaling might have therapeutic potential for
patients with ATLL. Either a CCR4 inhibitor or a PI3K
inhibitor might be considered (Pease and Horuk, 2014).
The anti-CCR4 monoclonal antibody KW-0761, which is
showing promising results in clinical trials (Yamamoto et al.,
2010; Ishida et al., 2012), was designed to promote antibody-
dependent cellular cytotoxicity of ATLL cells. This antibody
only inhibits chemotaxis weakly (Ishida et al., 2006), and its
effect on PI3K/AKT signaling has not been evaluated. To
improve the therapy of ATLL, our findings would support
the development of a therapeutic anti-CCR4 antibody
that both inhibits CCR4 signaling and mediates antibody-
dependent cellular cytotoxicity.
MATERIALS AND METHODS
Experimental design. All experiments presented have been repeated at
least two times, and consistent results were obtained. Data are depicted as
means SEM. Statistical comparisons were made using the Students t test.
P < 0.05 was considered statistically significant.
Patient samples and cell lines. Written informed consent was obtained in
accordance with the Declaration of Helsinki and was approved by the Investi-
gational Review Board of the National Cancer Institute (NCI). Some samples
were obtained before cytotoxic chemotherapy, whereas others were taken after
treatment (Table S1). PBMCs were isolated from ATLL patients by Ficoll-
Hypaque. In cases where the ATLL cells were <70% of the mononuclear cells,
the ATLL cells were purified by an initial negative selection magnetic column
method (Miltenyi Biotec) to enrich for CD4+ cells followed by a positive
selection on a CD25 column. The resultant population consisted of 7095%
CD4+CD25+ ATLL cells. The ATLL cell lines were provided by the follow-
ing researchers: M. Maeda (Kyoto University, Kyoto, Japan; ED40515(+),
ED40515(), ED41214(+), ED41214C(), ATL43T(+), ATL43Tb(),
ATL55T(+), and ATL42T(+)),Y.Yamada (Nagasaki University, Nagasaki, Japan;
ST1, KOB, KK1, and LMY1), T. Hata (Nagasaki University; ST1), T. Naoe
(Nagoya University, Nagoya, Japan; ATN1), and N. Arima (Kagoshima Uni-
versity, Kagoshima, Japan; Su9T01). 32D (a variant of the mouse 32D my-
eloid cell line engineered to express the human IL-2 receptor subunit) was
also used. IL2-dependent cell lines (ED40515(+), ED41214(+), ATL43T(+),
ATL55(+), ATL42T(+), KOB, KK, LMY1, and 32D) were cultured with
RPMI/10% FCS/penicillin-streptomycin with 100 IU/ml human recom-
binant IL2. Other lines were cultured with RPMI/10% FCS/penicillin-
streptomycin. ED40515(+) and KOB were engineered to express ecotropic
retroviral receptors and TET repressor for transduction of retroviral shRNA
vectors and expression vectors as previously described (Schmitz et al., 2012).
Reagents and antibodies. Doxycycline, puromycin, ethidium bromide, and
Lipofectamine 2000 were purchased from Invitrogen. Recombinant human
JEM Vol. 211, No. 13
Br ief Definitive Repor t
CCL17/TARC and recombinant human CCL22/MDC were purchased from
R&D Systems. Human recombinant IL2 was obtained from Roche. Anti
human CCR4 antibodies (clone 1G1) conjugated to PE and Alexa Fluor 647
fluors were purchased from BD. The following antibodies were purchased
from Cell Signaling Technology: Akt, P-Akt (Ser473; D9E), P-Akt (S473)
Alexa Fluor 647 (D9E), and Alexa Fluor 647conjugated isotype control
antibody (DA1E). Antirabbit IgG-HRP antibody was purchased from GE
Healthcare. CD8a(Lyt 2) microbeads were purchased from Miltenyi Biotec.
BKM120 was purchased from Selleck Chemicals. PTX was purchased from
Sigma-Aldrich. Big Dye Terminator v1.1 was purchased from Applied Biosys-
tems. Human gamma globulin fraction II was purchased from ICN Biomedicals
Inc. Paraformaldehyde was purchased from Electron Microscopy Sciences.
RNA-seq. RNA was extracted using the AllPrep kit (QIAGEN) and
sequencing libraries were prepared using the TruSeq RNA sample Prep kit v2
(Illumina) according to the manufacturers instructions. Paired-end 108-bp
read sequencing was performed on a HiSeq 2000 system (Illumina). The map-
ping of paired-end reads and the extraction of putative single nucleotide vari-
ants (SNVs) were performed as previously described (Schmitz et al., 2012). In
brief, paired-end reads were mapped to the RNA sequences in the RefSeq
database (NCBI build 37) using the Burrows-Wheeler Aligner (BWA) software
with default parameters. Reads that failed to map to RefSeq were mapped
to RNA sequences in the Ensembl database. The remaining unmapped reads
were mapped to the human genome assembly (NCBI build 37). Mutant SNV
calls were declared if more than two reads were mutated and the ratio of mu-
tant reads versus total coverage was >20%. SNVs that corresponded to single
nucleotide polymorphisms in the dbSNP database (build #132), the 1000
Genomes database (May 2011 release), and the NHLBI GO Exome Sequenc-
ing Project (ESP) database (ESP5400 December 2011 release) were excluded.
RNA-seq data were submitted to the NCBI Short Read Archive (SRA;
accession no. SRP042199).
Sanger DNA resequencing. Genomic DNA of CCR4 exon 2 region
was amplified by PCR using the primers CCR4-E2F, 5-CTTCCCCT
CATTAGCTGCTTCTGGTTG-3; and CCR4-E2R, 5-CCTGAC
ACTGGCTCAGGAATCTCTTAC-3. The purified PCR products were
sequenced using a Big Dye Terminator v1.1 cycle sequencing kit and
analyzed on an ABI 3730 Genetic Analyzer (Applied Biosystems). The
following primers were used for sequencing: CCR4-E2.1F, 5-CCTTC
CTGGCTTTCTGTTCAGCACTTG-3; and CCR4-E2.1R, 5-TGA
TTTCCAGGGAGCTGAGAACCTTCC-3.
Sanger RNA resequencing. RNA was DNase-digested and reverse tran-
scribed using the Omniscript RT kit (QIAGEN). The CCR4 coding region
was amplified by PCR using primers CCR4-E2F and CCR4-E2R. The
purified PCR products were cloned using the TOPO XL PCR Cloning kit
(Invitrogen). Sequencing was performed as described in the Sanger DNA
resequencing section.
CCR4 mutation search of public databases. cBioPortal and COSMIC
were used.
Retroviral vectors and retroviral transduction. CCR4-WT and CCR4-
Q330* cDNA was PCR amplified from ED40515(+) cDNA using the prim-
ers HindIII-CCR4-S, 5-GGAAGCTTTTGAAAGGCACCGGGTC-3;
and CCR4-TAG-BamHI-AS, 5-CGGGATCCCTACAGAGCATCATG-
GAGAT-3. The amplified fragments were cloned into HindIIBamHI sites
of doxycycline-inducible pRetroCMV/TO/puro and pRetroCMV/TO/PG
vectors. MSCV-CCR4-WT-ires-huKO or MSCV-CCR4-Q330*-ires-GFP
was made by inserting HindIII (blunted)XhoI fragments from pRetroCMV/
TO/CCR4-WT-puro or pRetroCMV/TO/CCR4-Q330*-puro vectors
into EcoRI(blunted)XhoI sites of MSCV-ires-huKO or MSCV-ires-GFP
(provided by S. Tsuzuki, Aichi Cancer Center Research Institute, Nagoya,
Japan). shRNA targeting the 3 UTR of CCR4 was cloned into doxycycline-
inducible pRSMX-puro vector with these two annealed oligos (shRNA target
2503
sequences are underlined): shCCR4-4A11_F, 5-GATCCCGAATGAAGTT-
GTAGGTAATTTCAAGAGAATTACCTACAACTTCATTCTTTTT-3;
and shCCR4-4A11_R, 5-AGCTAAAAAGAATGAAGTTGTAGGTA
ATTCTCTTGAAATTACCTACAACTTCATTCGG-3. pBMN-shCCR4-
ires-Lyt2 was made by insertion of XbaIBsmI fragment from pRSMX-
shCCR4-puro vector into XbaIBsmI sites of pBMN-ires-Lyt2 (provided by
G. Nolan, Stanford University, Stanford, CA). Retroviruses for ED40515(+)
and KOB were produced by cotransfection of 293T cells with helper plasmids
expressing gag-pol genes and an ecotropic pseudotyping env gene and a retro-
viral vector using Lipofectamine 2000 as described previously (Schmitz et al.,
2012). For 32D, retroviruses were produced by transfection of PlatE with ret-
roviral vectors. Target cells were infected with the retroviruses with 8 g/ml
polybrene with spin infection at 1,290 g for 1.5 h. pRetroCMV/TO-puro,
pRetroCMV/TO-PG, and pRSMX-infected cells was purified by using
2 g/ml puromycin. pBMN-ires-Lyt2infected cells were purified by MACS
mouse CD8a (Lyt2) microbeads (Miltenyi Biotech). Doxycycline was used at
40 ng/ml for induction of shRNA and cDNA.
Chemotaxis assay. HTS Transwell 96-well permeable supports with an
8.0-m pore polyester membrane (Corning) were used for chemotaxis assays
of ED40515(+) and KOB. 235 l of culture medium containing the indi-
cated dose of chemokine was added to the lower chamber, followed by the
addition of the Transwell inserts and the addition of cells in growth medium
(75 l) at a concentration of 5 105 cells/ml into the upper chamber. ChemoTx
(5 M pore; Neuro Probe) was used for chemotaxis assays of 32D. 300 l
of chemokine-containing medium was added to the lower chamber, and
55 l of cells in growth media (2 106 cells/ml) were added to the upper
chamber. After incubation for 2 h at 37C in 5% CO2, the upper chamber
was removed and 50 l of 1 PBS with a 1:100 dilution of SPHERO parti-
cles (SpheroTECH) and 1:2,500 dilution of 10 mg/ml ethidium bromide
was added into each lower chamber. The numbers of cells and SPHERO
particles in lower chamber were quantified with a FACSCalibur (BD), and
the numbers of cells that had migrated in each condition were normalized
based on the SPHERO particle count.
Flow cytometry for CCR4 surface staining. Cells were washed with
PBS containing 200 g/ml of human gamma globulin (Human Gamma
Globulin Fraction II; ICN Biomedicals Inc.) twice and incubated for 30 min
on ice with antihuman CCR4-PE or CCR4Alexa Fluor 647 (1G1; BD)
or control isotype IgG. After washing with FACS buffer (PBS/1% FCS)
twice, the cells were analyzed on a FACSCalibur (BD). For the CCR4 in-
ternalization assay, cells were exposed to CCL22 for varying lengths of time
and were then treated for 1 min at 37C to an acid buffer (pH 3.0, consisting
of 50 mM glycine and 100 mM NaCl) to remove cell-bound CCL22. The
cells were washed twice and then stained with antihuman CCR4Alexa
Fluor 647 on ice for 30 min, fixed with 2% paraformaldehyde (Electron
Microscopy Sciences) on ice for 10 min, and analyzed on a FACSCalibur.
Immunoblot analysis. Cells were washed and resuspended in 2 SDS
sample buffer at 106 cells/100 l and boiled for 5 min. Samples were sepa-
rated on Novex 412% Tris-Glycine gel (Invitrogen) and transferred to a
PVDF membrane (Immobilon-P; EMD Millipore). Proteins were detected
with the following primary antibodies: p-AKT(S473) (D9E) and AKT (5G3;
Cell Signaling Technology). An antirabbit IgG-HRP antibody (GE Health-
care) was used as the secondary antibody.
AKT phospho-flow analysis. After incubation with 50 ng/ml CCL22 for
the indicated time, cells were fixed in 2% paraformaldehyde for 10 min at
room temperature and then permeabilized with cold methanol at 20C
overnight. Cells were washed twice in FACS buffer and stained with p-Akt
(S473)Alexa Fluor 647 (D9E) or isotype control (DA1E; Cell Signaling
Technology) for 20 min at room temperature. Cells were washed and resus-
pended in FACS buffer for analysis on a FACSCalibur.
Online supplemental material. Table S1, included as a separate Excel
file, shows Sanger sequencing analysis of exon 2 of CCR4 (NM_005508.4)
2504
in 53 cases of ATLL samples. Online supplemental material is available at
http://www.jem.org/cgi/content/full/jem.20140987/DC1.
We thank the following researchers for providing ATLL cell lines: Michiyuki Maeda,
Yasuaki Yamada, Tomoko Hata, Tomoki Naoe, and Naomichi Arima. We also thank
Richard N. Bamford, Liyanage P. Perera, Ryan Young, Art Shaffer, and Michele
Ceribelli for helpful discussions.
This research was supported by the Intramural Research Program of the
National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Roland Schmitz was supported by the Dr. Mildred Scheel Stiftung fr
Krebsforschung (Deutsche Krebshilfe).
The authors declare no competing financial interests.
Submitted: 23 May 2014
Accepted: 13 November 2014
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2505
 Brief Definitive Report

Enhancement of an anti-tumor immune

response by transient blockade
of central T cell tolerance
Imran S. Khan,1 Maria L. Mouchess,1 Meng-Lei Zhu,3,4 Bridget Conley,3,4
Kayla J. Fasano,1 Yafei Hou,2 Lawrence Fong,2 Maureen A. Su,3,4
and Mark S. Anderson1
1DiabetesCenter and 2Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco,
San Francisco, CA 94143
3Department of Pediatrics and 4Department of Microbiology/Immunology, School of Medicine, and Lineberger Comprehensive

Cancer Center, University of North Carolina, Chapel Hill, NC 27599

Thymic central tolerance is a critical process that prevents autoimmunity but also presents
a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial
cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-
specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against
autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells
by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on
autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to
deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of
tumor-specific effector T cells. Recent work has implicated the TNF family members RANK
and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in
vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the
thymus to create a window of defective negative selection. Furthermore, we demonstrate
that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that
persistence of these tumor-specific effector T cells promoted increased host survival in
response to tumor challenge. These results indicate that modulating central tolerance
through RANKL can alter thymic output and potentially provide therapeutic benefit by
enhancing anti-tumor immunity.

CORRESPONDENCE
Mark S. Anderson:
manderson@diabetes.ucsf.edu
Abbreviations used: Aire,
autoimmune regulator; cTEC,
cortical thymic epithelial cell;
IRBP, interphotoreceptor
retinoid-binding protein; mTEC,
medullary thymic epithelial cell;
OPG, Osteoprotegerin; TSA,
tissue-specific antigen.
Medullary thymic epithelial cells (mTECs) con-
tribute to self-tolerance through the ectopic ex-
pression of tissue-specific antigens (TSAs) in the
thymus (Derbinski et al., 2001; Anderson et al.,
2002; Metzger and Anderson, 2011). This TSA
expression in mTECs is largely dependent on
autoimmune regulator (Aire), which is expressed
in mature mTECs (Gbler et al., 2007; Gray et al.,
2007; Metzger and Anderson, 2011). Through
the recognition of TSAs, developing autoreactive
T cells are either negatively selected from the pool
of developing thymocytes or recruited into the
regulatory T (T reg) cell lineage (Liston et al., 2003;
Anderson et al., 2005; DeVoss et al., 2006; Shum
et al., 2009; Taniguchi et al., 2012; Malchow et al.,
2013). The overall importance of this process is
underscored by the development of a multi-organ
I.S. Khan and M.L. Mouchess contributed equally to
this paper.
autoimmune syndrome in patients or mice with
defective AIRE expression (Consortium, 1997;
Nagamine et al., 1997; Anderson et al., 2002).
Although central tolerance provides pro-
tection against autoimmunity, this process also
represents a challenge for anti-tumor immunity
(Kyewski and Klein, 2006; Malchow et al., 2013).
Because many of the TSAs expressed in the thy-
mus are also expressed in tumors, high-affinity
effector T cells capable of recognizing tumor
self-antigens may normally be deleted in the thy-
mus (Bos et al., 2005; Cloosen et al., 2007; Trger
et al., 2012; Zhu et al., 2013). Transiently sup-
pressing central tolerance by depleting mTECs
or modulating Aire expression may provide a
2014 Khan et al. This article is distributed under the terms of an Attribution
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J. Exp. Med. 2014 Vol. 211 No. 5 761-768 761
www.jem.org/cgi/doi/10.1084/jem.20131889

therapeutic window for the generation of T cells capable of
recognizing tumor self-antigens. Many current cancer immune
therapies rely on activating relatively weak tumor-specific T cell
responses through modulating peripheral tolerance (Swann and
Smyth, 2007; Chen and Mellman, 2013). In contrast, manipu
lation of central tolerance has the potential to increase the
pool and affinity of effector T cells that can recognize and con-
tribute to effective anti-tumor responses. Furthermore, such
high-affinity, self-reactive T cells may be more resistant to pe-
ripheral tolerance mechanisms that typically restrain an anti-
tumor response (Swann and Smyth, 2007).Thus, the development
of methods that selectively and transiently deplete Aire-expressing
mTECs may be an attractive method to enhance tumor-specific
immune responses.
Previous work has identified agents that can inhibit the
growth and development of TECs such as corticosteroids, cy-
closporine, and some inflammatory cytokines (Anz et al., 2009;
Fletcher et al., 2009). Despite their clear inhibitory effects on
TECs, however, these agents do not appear to have selectivity
for blocking mTEC development. Interestingly, recent studies
have demonstrated a role for TNF family member pairs
RANKRANKL and CD40-CD40L in the embryological
development of Aire+ mTECs (Rossi et al., 2007; Akiyama et al.,
2008; Hikosaka et al., 2008; Roberts et al., 2012). Recent work
762
Figure 1. Selective depletion of mTECs with anti-
RANKL blockade. (A) Wild-type mice were treated for 2 wk
with either isotype (gray) or anti-RANKL (RANKL, blue)
antibody, and absolute numbers of mTECs and cTECs were
enumerated by flow cytometry. Bar graphs of total cell num-
bers are depicted by mean SEM. (B) Representative flow
cytometry plots of mTECs in A showing relative composition
of mTEC subsets. Values represent mean SEM. Bar graphs
(right) of total cell numbers of each mTEC subset depicted by
mean SEM. (C) Top panels show immunostaining for keratin-8
(green) and keratin-5 (red) on thymic sections, and bottom
panels show immunostaining for keratin-5 (red) and
Aire (green). Bars: (top) 500 m; (bottom) 50 m. (D) Gene
expression analysis on mTECs sorted from wild-type mice
treated with either isotype (gray) or anti-RANKL (blue) anti-
body. Results standardized to Cyclophilin A and normalized
to isotype-treated mice with bars depicting mean SD.
(E) Representative flow cytometry plots of thymocytes from
antibody-treated wild-type mice. Values depict mean SD.
Total thymocyte numbers (right) are indicated with each
circle depicting an individual animal and bars showing the
mean. (F) Flow cytometric analysis of Foxp3 staining in CD4
SP thymocytes from E. Plots (right) show percentage and
number of thymic CD4+ Foxp3+ cells, with bars showing the
mean. Data shown in Fig. 1 is representative of two to four
independent experiments containing three or more indi-
vidual mice within each group. *, P 0.05; ***, P 0.001,
Students t test.
has also demonstrated that mTECs in particular have a rela-
tively fast turnover in adult mice with an estimated half-life of
2 wk (Gbler et al., 2007; Gray et al., 2007). Given these find-
ings, we speculated that in vivo blockade of RANKL in adult
hosts could both selectively and transiently inhibit the develop-
ment and turnover of mTECs with potential to alter central
T cell tolerance. To this end, we performed in vivo RANKL
blockade in adult mice and investigated its effects on both TECs
and developing thymocytes.We show that anti-RANKL treat-
ment not only depleted mTECs but could also be used thera-
peutically to break central tolerance and, as a result, increase
the generation of tumor-specific T cells.
RESULTS AND DISCUSSION
Depletion of mTECs with RANKL blockade
The RANKRANKL signaling pathway is important for
mTEC development, but it remains unclear what impact per-
turbation of this pathway might have on the adult thymus.
Previous work has linked the RANKRANKL pathway to
the development of Aire-expressing mTECs (Rossi et al.,
2007; Akiyama et al., 2008; Hikosaka et al., 2008; Roberts
et al., 2012), and we hypothesized that treating mice with
blocking anti-RANKL antibody would decrease Aire+ mTECs.
RANKL blockade enhances anti-tumor immunity | Khan et al.
 Br ief Definitive Repor t

We treated wild-type mice with either anti-RANKL or isotype

control antibody for 2 wk and harvested their thymi for analy
sis. Interestingly, although mice treated with anti-RANKL
showed only a modest loss of cortical thymic epithelial cell
(cTEC) cellularity, they exhibited a severe depletion of >80%
of mTECs (Fig. 1 A). Using MHC II and Aire as markers of
mTEC maturation (Gbler et al., 2007; Gray et al., 2007), we
further analyzed the immature mTEClo (MHC IIlo Aire),
intermediate mTEChi (MHC IIhi Aire), and mature Aire+
mTEC subsets (MHC IIhi Aire+).The relative mTEC compo-
sition in anti-RANKLtreated mice revealed a substantial loss
of Aire+ and mTEChi cells along with enrichment of the re-
maining mTEClo cells (Fig. 1 B). Although absolute numbers
of mTECs were decreased across all mTEC subsets, mTEC
depletion was mostly due to the loss of >90% of all Aire+ and
mTEChi cells (Fig. 1 B). Immunostaining revealed a slight de-
crease in the density of keratin-5+ (K5) cells in the medulla of
anti-RANKLtreated mice, whereas Aire+ cells were nearly
undetectable (Fig. 1 C). Importantly, staining for keratin-8
(K8) and K5 showed that the overall corticomedullary thymic
architecture was preserved despite the loss of mature mTECs
Figure 2. Increased Aire+ mTECs in OPG/ mice. (A) Quantitative
(Fig. 1 C). Furthermore, consistent with the loss of the Aire+ PCR analysis of Aire and OPG gene expression in MHC IIlo and MHC IIhi
and mTEChi subsets, Aire-dependent TSA gene expression in mTECs sorted from wild-type B6 mice. Results standardized to Cyclophilin
sorted mTECs was decreased in anti-RANKLtreated mice A and normalized to MHC IIlo, with error bars depicting mean SD.
(Fig. 1 D). (B) Absolute numbers of mTECs and cTECs in OPG+/+ (black) and OPG/
Next, we characterized the impact of anti-RANKLmedi- (red) mice were enumerated by flow cytometry. Bar graphs of total cell
ated mTEC depletion on thymocyte selection. In the poly- numbers are depicted by mean SEM. (C) Representative flow cytometry
clonal T cell repertoire of wild-type mice treated with anti- plots of mTECs in B showing relative composition of indicated mTEC sub-
RANKL, we observed a modest increase in frequencies of sets. Values depict mean SEM. Bar graphs (right) depict total cell num-
bers of each mTEC subset and indicate mean SEM. (D) Immunostaining
both CD4 single-positive (SP) and CD8 SP thymocytes, con-
for keratin-8 (green) and keratin-5 (red) on frozen thymic sections from
sistent with a lack of negative selection (Fig. 1 E). Importantly, OPG+/+ and OPG/ mice. Data are representative of at least three inde-
anti-RANKLtreated mice showed only a slight reduction in pendent experiments with at least three mice per group. *, P 0.05;
the frequency of double-positive thymocytes while absolute **, P 0.01; ***, P 0.001, Students t test. Bars, 500 m.
numbers were maintained, confirming normal positive selec-
tion. In addition, total thymocyte numbers were also maintained
in mice treated with anti-RANKL (Fig. 1 E). Furthermore, corticomedullary thymic architecture despite these changes
anti-RANKLtreated mice showed a 50% reduction of Foxp3+ in TEC cellularity and mTEC composition (Fig. 2 D).
T reg cells within the CD4 SP subset (Fig. 1 F). Collectively, these data suggest that RANKRANKL
Given the dramatic impact of RANKL blockade on OPG interactions regulate both TEC cellularity and mTEC
mTECs, we sought to determine whether this effect could be maturation, and are particularly important for the induction of
reversed in the context of increased RANK signaling. Osteo- Aire+ mTECs. Furthermore, anti-RANKL treatment selec-
protegerin (OPG; Tnfrsf11b) is a soluble decoy receptor for tively targeted mature mTECs and altered negative selection
RANKL and its role as a negative regulator of RANK signal- without affecting the development of new thymocytes.
ing has been well described in bone physiology (Kearns et al.,
2008). Interestingly, in sorted wild-type mTECs, OPG ex- Regeneration of mTECs after withdrawal of anti-RANKL
pression was up-regulated in MHC IIhi mTECs when com- We next examined the kinetics of mTEC recovery after with-
pared with MHC IIlo mTECs (Fig. 2 A). To test whether drawal from anti-RANKL blockade. After a 2-wk treatment
OPG negatively regulates RANKRANKL signaling in window, we harvested mice at 2-wk intervals and observed
mTECs, we analyzed thymi from OPG/ mice.While cTEC gradual recovery of the mTEChi and Aire+ subsets (Fig. 3,
cellularity was increased threefold in OPG/ mice, mTEC A and B). By 10 wk, we observed the complete recovery of
cellularity was increased by nearly 10-fold when compared all mTEC subsets and a normal level of Aire expression after
with OPG+/+ mice (Fig. 2 B). Although absolute numbers of anti-RANKL treatment (Fig. 3 B). Interestingly, the pattern of
all mTEC subsets were increased in OPG/ mice, we ob- mTEC recovery appeared consistent with published reports
served an enrichment of Aire+ mTECs and a proportional of mTEClo cells giving rise to mTEChi cells before the induction
loss of the mTEClo subset (Fig. 2 C). Immunostaining of of Aire+ mTECs (Fig. 3 B; Gbler et al., 2007; Gray et al., 2007;
K8 and K5 showed that OPG/ thymi maintained their Rossi et al., 2007). Notably, thymi from anti-RANKL and

JEM Vol. 211, No. 5 763


isotype-treated mice at 10 wk were indistinguishable by im-
munostaining (Fig. 3 C). Thus, anti-RANKLmediated mTEC
depletion is a transient phenomenon with return of normal
thymic composition after withdrawal of antibody treatment.
Manipulation of thymic negative selection
with RANKL blockade
To further characterize the impact of anti-RANKL treatment
on negative selection, we first used the OT-II CD4+ TCR
x RIP-mOVA double transgenic mouse model. RIP-mOVA
764
Figure 3. Regeneration of mTECs after anti-RANKL with-
drawal. (A) Experimental layout for analysis of mTEC recovery
after anti-RANKL withdrawal. Wild-type mice were treated for
1 wk with isotype or anti-RANKL (black arrows) and harvested at
indicated time points (red arrows). Graph (bottom) depicts rela-
tive composition of mTEClo mTEChi and Aire+ mTECs at each time
point in anti-RANKLtreated mice. Values represent mean
SEM. (B) Representative flow cytometry plot of mTECs from
each of the indicated time points from A. Values represent the
frequency of Aire+ mTECs. (C) Immunostaining for keratin-8
(green) and keratin-5 (red) on thymic sections from isotype or
anti-RANKLtreated mice harvested at 10 wk. Bars, 500 m.
Data shown is representative of at least two independent experi-
ments with three to five mice per group.
transgenic mice express a membrane form of OVA under the
control of the rat insulin promoter which results in its expres-
sion in both the pancreatic islets and in mTECs (Anderson et al.,
2005). When the OT-II CD4+ TCR transgenic line is crossed
to the RIP-mOVA transgenic line, OVA-specific OT-II T cells
are deleted in the thymus (Anderson et al., 2005). We treated
both OT-II and OT-II x RIP-mOVA mice with either anti-
RANKL or isotype control antibody and performed thymocyte
analysis. Consistent with previous reports, OT-II x RIP-mOVA
mice treated with control antibody showed a significant
Figure 4. Anti-RANKLmediated mTEC
ablation leads to altered negative selection.
(A) OT-II and OT-II x RIP-mOVA mice were har-
vested after 2 wk of indicated antibody treat-
ment. Percentages of CD4 SP (top) and OT-II
clonotype-specific (bottom) thymocytes are
shown. Values represent mean SD. (B) Quanti-
fication of OT-II clonotype-specific CD4 SP cells
from A. Graph shows mean SEM. **, P 0.01,
Students t test. (C) Representative histograms
for Foxp3 staining in CD4 SP events from A.
(D) Wild-type mice were treated with isotype or
anti-RANKL antibody for 3 wk, immunized with
P2 peptide, and then harvested 10 d later for
tetramer analysis. Representative flow cytom-
etry plots of CD4+ T cells are shown for each
condition. Values indicate absolute numbers of
CD44+ P2-I-Abspecific T cells. IRBP/ mice
were included as positive controls. Each dot on
the graph represents an individual mouse, bars
show mean, and the dotted line represents the
limit of detection. Data shown is representative
of two to three independent experiments.
*, P 0.05, Mann-Whitney test.
RANKL blockade enhances anti-tumor immunity | Khan et al.

represent mean SEM. ***, P 0.001, Students t test. Data shown is n = 79 mice per group pooled from two independent experiments. (G) Survival curves
of RAG1/ recipients inoculated with B16 melanoma after adoptive transfer of splenocytes from either anti-RANKL (blue) or isotype (gray)treated
RAG1/ x TRP-1 TCR Tg mice, n = 10 mice for each group. P 0.001, Log-rank test. Data shown is representative of two independent experiments.
reduction in the proportions of CD4 SP thymocytes and also
had decreased frequencies and numbers of OT-II+ T cells (Fig. 4,
A and B; Anderson et al., 2005). In contrast, thymocyte pro-
files of anti-RANKLtreated OT-II x RIP-mOVA mice were
indistinguishable from that of OT-II mice, demonstrating that
RANKL blockade prevented thymic deletion of OT-II T cells
yet allowed their positive selection (Fig. 4, A and B). We also
observed a loss of thymic T reg cell development in these
mice, which suggested a loss of cognate antigen (OVA) ex-
pression from mTECs within the thymus (Fig. 4 C).
To expand these observations to the polyclonal T cell rep-
ertoire, we analyzed the development of Aire-dependent auto-
reactive T cells using a tetramer enrichment protocol. Previously,
we had shown that T cells specific for the self-antigen inter-
photoreceptor retinoid-binding protein (IRBP) are deleted in
JEM Vol. 211, No. 5
Br ief Definitive Repor t
Figure 5. RANKL blockade increases
anti-melanoma immune response.
(A and B) RAG1/ x TRP-1 TCR Tg mice were
treated with isotype or anti-RANKL antibody
for 2 wk and thymus and spleen were har-
vested for analysis. Flow cytometry plots in A
show percentage of CD4+ (top) and CD4-gated
V14+ (bottom) T cells. Bar graphs in B show
quantification of data in A. Values represent
mean SEM. *, P 0.05; **, P 0.01;
***, P 0.001, Students t test. Data shown is
representative of at least three independent
experiments with n = 47 mice per group.
(C) Ear skin from mice treated with isotype (gray)
or anti-RANKL (blue) antibody was analyzed
by qPCR for Tyrosinase and Tyrp1 expression.
Results standardized to 2m and normalized
to untreated B6 wild-type mice (black) with
bars depicting mean SD. (D) Survival curves
of RAG1/ x TRP-1 TCR Tg mice inoculated
with B16 melanoma after treatment with iso-
type (gray) or anti-RANKL (blue) antibody,
n = 78 mice for each group. P 0.05, Log-rank
test. Data shown is representative of four inde-
pendent experiments. (E) Tumor sections from
isotype or anti-RANKLtreated mice in D were
immunostained for CD3 (pink) with DAPI coun-
terstain (purple). Bars, 50 m. Mean densities
of CD3+ cells per tumor area are quantified on
the right. Four sections were evaluated for
each treatment group and 6 imaging fields
were randomly scored from each section.
Graphs depict mean SEM. *, P 0.05, Stu-
dents t test. (F) RAG1/ x TRP-1 TCR Tg mice
were inoculated with B16 melanoma after
treatment with isotype (gray) or anti-RANKL
(blue) antibody. Mice were harvested at 21 d
after tumor inoculation and tumor-infiltrating
lymphocytes (TILs) were analyzed by flow cy-
tometry. Graph depicts percentage of Foxp3+
CD25+ T reg cells among CD4+ T cells. Values
thymus of Aire+/+ mice, whereas these cells escape deletion
in Aire/ mice and cause autoimmune uveitis (DeVoss et al.,
2006; Taniguchi et al., 2012). Through the use of an IRBP pep-
tide class II tetramer, P2-I-Ab, such autoreactive CD4+ T cells
can be detected in Aire/ mice. Given both the severe deple-
tion of Aire+ mTECs and the loss of thymic IRBP expression
in anti-RANKLtreated mice, we hypothesized that P2-I-Ab
specific T cells could be detected in treated mice. After treating
wild-type mice with anti-RANKL antibody, we immunized
mice with a MHC IIbinding IRBP peptide epitope (P2) to
expand T cells for detection. 10 d after immunization, lymph
nodes and spleen were pooled for the enumeration of CD4+
P2-I-Abspecific T cells by flow cytometry. Consistent with the
loss of Aire+ mTECs, tetramer analysis of anti-RANKLtreated
mice revealed an expansion of CD4+ P2-I-Abspecific T cells
765
(Fig. 4 D). Overall, these data show a clear defect in negative
selection associated with the loss of Aire+ mTECs in anti-
RANKLtreated mice.
Treatment with anti-RANKL enhances anti-tumor immunity
Given its ability to selectively block mTECs and central toler-
ance, we next sought to determine whether anti-RANKL
treatment could be used to therapeutically enhance anti-tumor
immunity. Previous work has shown that many tumor self-
antigens are expressed by mTECs as TSAs, and that high-affinity
T cells capable of recognizing these tumor self-antigens are ef-
ficiently deleted in the thymus (Bos et al., 2005; Trger et al.,
2012; Zhu et al., 2013). To test whether anti-RANKL treat-
ment could rescue tumor self-antigenspecific T cells from
thymic deletion, we used the TRP-1 CD4+ TCR transgenic
mouse model which generates T cells specific for the mela-
noma antigen TRP-1 (Tyrp1; Muranski et al., 2008). TRP-1 is
expressed in B16 melanoma cells, and TRP-1 T cells are effica-
cious against established B16 melanoma tumors (Muranski et al.,
2008). Importantly, mTECs endogenously express TRP-1 in an
Aire-dependent manner, such that TRP-1specific T cells are
deleted in the thymi of Aire-sufficient, TRP-1sufficient hosts
(Muranski et al., 2008; Zhu et al., 2013). Given the depletion of
Aire+ mTECs and loss of thymic TRP-1 expression in anti-
RANKLtreated mice (Fig. 1 D), we hypothesized that anti-
RANKL treatment could rescue TRP-1 T cells from thymic
deletion. We treated RAG1/ x TRP-1 TCR transgenic mice
with either anti-RANKL or control antibody and harvested
their thymi and spleens for analysis. Consistent with previous
reports, isotype-treated mice showed efficient deletion of CD4
SP T cells in the thymus (Fig. 5 A; Muranski et al., 2008; Zhu
et al., 2013). In contrast, anti-RANKL treatment prevented
deletion of CD4 SP cells and also resulted in a much higher
percentage of V14+ T cells (Fig. 5, A and B). Furthermore,
TRP-1 T cells were detectable in the spleens of anti-RANKL
treated mice and also expressed higher levels of the V14 TCR
(Fig. 5, A and B). In addition, qPCR analysis revealed a loss of
Tyrosinase and Tyrp1 expression in the ear skin of anti-RANKL
treated mice which appeared consistent with the destruction of
melanocytes by TRP-1 T cells (Fig. 5 C).
Next, we challenged anti-RANKLtreated RAG1/ x
TRP-1 mice with B16 melanoma to determine whether a lim-
ited break in central tolerance could improve overall survival.
We observed a statistically significant increase in the survival of
anti-RANKLtreated mice compared with the isotype-treated
cohort (Fig. 5 D).We also found evidence of an enhanced T cell
response in anti-RANKLtreated mice by measuring CD3+
T cell infiltrates within the tumors of these mice (Fig. 5 E). Of
note, given the overall increase in T reg cells in the spleen and
tumors of anti-RANKLtreated mice, it appears unlikely that
the survival benefit observed in TRP-1 mice is due to differ-
ences in T reg cell numbers (Fig. 5, B and F). Furthermore, to
exclude potential effects of anti-RANKL antibody on the
tumor microenvironment, we performed adoptive transfer
studies in which splenocytes from antibody-treated RAG1/
x TRP-1 mice were transferred into RAG1/ mice. When
766
challenged with B16 melanoma, recipients of splenocytes from
anti-RANKLtreated donors again showed a statistically sig-
nificant increase in survival (Fig. 5 G). Collectively, these data
demonstrate that short-term, reversible RANKL blockade of
mTEC development can be used to create a therapeutic win-
dow that allows tumor self-antigenspecific T cells to escape
thymic deletion. Furthermore, the generation of these high-
affinity tumor-specific T cells confers a survival benefit in a
melanoma tumor model.
In conclusion, our findings provide strong evidence that
mTECs can be selectively and therapeutically targeted by
RANKL blockade in adult mice and that anti-RANKL can
be used as an approach to enhance anti-tumor responses. To
date, previous efforts on the therapeutic manipulation of
TECs have shown global effects that involve both cTECs and
mTECs and disrupt thymocyte development (Fletcher et al.,
2009). The selectivity for mTECs with anti-RANKL thus
provides evidence that central tolerance can be transiently sup-
pressed in adult hosts while maintaining T cell generation. Al-
though autoimmunity is a dangerous potential consequence
of this approach, the treatment may also be an attractive new
method to help break tolerance for cancer immunotherapy.
Interestingly, although we could detect an expansion of
retina-specific T cells in anti-RANKLtreated mice, we did
not observe development of spontaneous uveitis (unpublished
data), suggesting that this brief window of central tolerance
suppression was countered by peripheral tolerance mecha-
nisms such as T reg cells which existed before treatment. Im-
portantly, we find that the medullary epithelial compartment
of the thymus recovers after withdrawal of anti-RANKL an-
tibody and, thus, only a transient window of central tolerance
suppression occurs. This window may be an attractive feature
of this approach, especially if coupled with methods that pref-
erentially expand tumor-specific T cells over pathogenic auto-
reactive T cells. Currently, there has been intense interest and
progress in manipulating peripheral tolerance for immuno
therapy (Chen and Mellman, 2013). Given our results, it will
also be of interest to determine if a combination of methods
that target both central and peripheral tolerance could fur-
ther enhance anti-tumor immune responses. Finally, it is impor-
tant to note that our results also have implications for patients
in the clinical setting receiving denosumab, a humanized mono-
clonal antibody that blocks RANKL which is widely used in
the treatment of osteoporosis in adults (Cummings et al., 2009).
Further study will be needed in such patients regarding their
susceptibility to autoimmunity and for other potential defects
in central tolerance.
MATERIALS AND METHODS
Mice. C57BL/6, B6.RAG1/ Tyrp1B-w TRP-1 TCR transgenic, and B6.
OPG/ mice were purchased from The Jackson Laboratory. Mice were
treated intraperitoneally with 100 g anti-RANKL (IK22/5) or isotype con-
trol (2A3; Bio X Cell) antibody three times per week as stated in the text.
B6.OT-II, B6.RIP-mOVA, and B6.IRBP/ mice were described previously
(Anderson et al., 2005; DeVoss et al., 2006). Mice were treated at 35 wk of
age and harvested at time points indicated in the text. All mice were housed
and bred in specific pathogen-free conditions in animal facilities at UCSF or
RANKL blockade enhances anti-tumor immunity | Khan et al.

UNC, Chapel Hill. Animal experiments were approved by the Institutional
Animal Care and Use Committee (IACUC) at UCSF or UNC, Chapel Hill.
Quantitative PCR. RNA was extracted from sorted mTECs using the
RNeasy Micro Plus kit (QIAGEN) and cDNA was synthesized with the In-
vitrogen Superscript III kit. TaqMan gene expression assays (Applied Biosys-
tems) were used for all targets.
Histology and immunofluorescence. Thymi were harvested and embed-
ded in O.C.T. media (Tissue-Tek). 8-m frozen thymic sections were fixed in
100% acetone, blocked, and stained for keratin-5, keratin-8 (Abcam), or Aire
(eBioscience). Secondary antibodies were purchased from Invitrogen. Thymic
sections were visualized using a widefield microscope (Apotome; Carl Zeiss).
For tumor immunostaining, tumors were harvested and fixed in 10% formalin
as previously described (Zhu et al., 2013). Fixed tumors were embedded in
paraffin and sectioned for staining with anti-CD3 antibody and counterstained
with DAPI. Tumor sections were visualized using a fluorescent microscope
(BX60; Olympus) and analyzed using ImageJ software (National Institutes
of Health).
Flow cytometry. TECs were isolated as previously described (Gardner
et al., 2008). In brief, thymi were minced and digested with DNase I and
Liberase TM (Roche) before gradient centrifugation with Percoll PLUS (GE
Healthcare). Enriched stromal cells were stained with the indicated surface
marker antibodies (BioLegend). mTECs were defined as CD45, EpCAM+,
MHC II+, Ly51 events and cTECs were defined as CD45, EpCAM+,
MHC II+, Ly51+ events. For lymphocyte staining, all surface marker anti-
bodies were obtained from BioLegend. For intracellular staining, cells were
stained using the Foxp3 Staining Buffer Set and stained with anti-Foxp3
or anti-Aire (eBioscience). All data were collected using a flow cytometer
(LSR II; BD) and analyzed with either FlowJo software (Tree Star) or FACS
Diva (BD). Cell sorting was performed using a FACS Aria III cell sorter (BD).
IRBP P2 peptide immunization and tetramer analysis. Mice were
immunized with 100 g P2 peptide (IRBP; aa 271290) emulsified in com-
plete Freunds adjuvant as described previously (Taniguchi et al., 2012).
Tetramer analysis was performed on pooled lymph nodes and spleen from
treated mice 10 d after immunization. P2-I-Ab tetramer was generated by the
National Institutes of Health Tetramer Core Facility, and tetramer staining
was performed as described previously (Taniguchi et al., 2012). After tetramer
enrichment, cells were stained with antibodies for flow cytometry, and count-
ing beads (Invitrogen) were used to enumerate tetramer+ cells.
B16 melanoma tumor challenge. B6.RAG1/TRP-1 TCR transgenic
mice were injected subcutaneously in the left flank with 1.0 105 B16 mela-
noma cells. For adoptive transfer studies, spleens from donor mice were
pooled and CD25-depleted before transfer into B6.RAG1/ hosts. Recipi-
ent mice were inoculated with 7.5 104 B16 melanoma cells 7 d after trans-
fer. Tumor growth was monitored by taking measurements of length (L) and
width (W), and tumor volume was calculated as LW2/2. For generation of
survival curves, death was defined as tumor size >1,000 mm3.
Statistical analysis. Statistical analysis was performed using Prism 6.0
(GraphPad Software). Mann-Whitney Rank sum testing was performed on
tetramer analysis. Students t test was performed for TEC and lymphocyte
analyses. Log-rank test was performed for Kaplan-Meier survival curves.
We thank T. Metzger, T. LaFlam, M. Cheng, and W. Purtha for critical reading of the
manuscript. We thank the National Institutes of Health Tetramer Core Facility for
providing tetramer reagent.
This work was supported by the US National Institutes of Health Grants
AI097457 (M.S. Anderson) and K12-GM081266 (M.L. Mouchess), and the UCSF
Medical Scientist Training Program (I.S. Khan). Flow cytometry data were generated
in the UCSF Parnassus Flow Cytometry Core, which is supported by the Diabetes
and Endocrinology Research Center (DERC) grant NIH P30 DK063720.
The authors declare no competing financial interests.
JEM Vol. 211, No. 5
Br ief Definitive Repor t
Submitted: 6 September 2013
Accepted: 3 April 2014
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RANKL blockade enhances anti-tumor immunity | Khan et al.
 Brief Definitive Report

T cellderived interleukin (IL)-21 promotes

brain injury following stroke in mice
Benjamin D.S. Clarkson,1,3 Changying Ling,1 Yejie Shi,2 Melissa G. Harris,1,4
Aditya Rayasam,4 Dandan Sun,2,5 M. Shahriar Salamat,1 Vijay Kuchroo,6
John D. Lambris,7 Matyas Sandor,1 and Zsuzsanna Fabry1
1Department of Pathology and Laboratory Medicine, 2Department of Neurological Surgery, 3Department of Cellular
and Molecular Pathology, 4Neuroscience Training Program, School of Medicine and Public Health, University
of Wisconsin-Madison, Madison, WI 53792
5Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, PA 15213
6Center for Neurological Diseases, Brigham and Womens Hospital Harvard Medical School, Boston, MA 02115
7Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion
injury, but the relevant T cellderived mediators of tissue injury remain unknown. Using a
mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated
in the injured mouse brain after cerebral ischemia. IL-21deficient mice have smaller
infarcts, improved neurological function, and reduced lymphocyte accumulation in the
brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer
experiments revealed that brain-infiltrating CD4+ T cells are the predominant IL-21 source.
Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion
injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4+ T cells in the
area surrounding acute stroke lesions, suggesting that IL-21mediated brain injury may be
relevant to human stroke.

CORRESPONDENCE
Zsuzsanna Fabry:
zfabry@facstaff.wisc.edu
Abbreviations used: BA, basilar
artery; ECA, external carotid
artery; ICA, internal carotid
artery; I/R, ischemia/reperfu-
sion; MCA, middle cerebral
artery; PCA, posterior cerebral
artery; PComA, posterior com-
municating artery; RAG,
recombination activating
gene; rCBF, regional cerebral
blood flow; tMCAO, transient
MCA occlusion; TTC, 2,3,5-
triphenyltetrazolium chloride;
VA, vertebral artery.
Stroke is one of the leading causes of death and
disability worldwide. Clinical and preclinical ex-
perimental studies highlight the importance of
inflammation in both acute and delayed neuro-
nal tissue damage after ischemic stroke; however,
the mechanisms and cells involved in this neuro-
inflammation are not fully understood. There is
currently no available treatment targeting the
acute immune response that develops in the
brain after transient focal ischemia. Therefore,
we sought to identify novel T cellderived cyto-
kines that contribute to acute cerebral reperfu-
sion using the mouse model of transient middle
cerebral artery occlusion (tMCAO).
During the reperfusion of infarcted brain tis-
sue, leukocytes accumulate in the injured brain
where, in addition to clearing cell debris, they
promote secondary tissue injury (Yilmaz and
Granger, 2010). Within the acute phase of isch-
emic reperfusion (I/R) injury there are multiple
waves of cell infiltration of macrophages, neutro-
phils, and lymphocytes (Gelderblom et al., 2009).
Brain-infiltrating T cells have also been widely
reported in stroke and animal models of stroke
and are thought to have acute detrimental and
delayed protective effects (Magnus et al., 2012).
Conventionally, the protective role of T cells has
been attributed to the accumulation of regula-
tory T cells within the CNS in later stages of re-
perfusion injury. These T cells produce a variety
of cytokines including TGF and IL-10, which
are both antiinflammatory and neuroprotective.
(Liesz et al., 2009; Stubbe et al., 2013). In addi-
tion to having an established role in delayed
neuroprotection, Kleinschnitz et al. (2013) have
recently shown that CD4+ CD25+ regulatory
T cells also promote acute ischemic injury through
interaction with the cerebral vasculature. The
acute detrimental effects can be further divided
into early (24 h) and late (72 h) phases, with
IL-17 production by nonconventional T cells
(less common T cell subset associated with mu-
cosal tissues) possibly accounting for the latter by
promoting neutrophil accumulation (Gelderblom
et al., 2012).
The mechanisms of the early detrimental ef-
fects of T cells after cerebral ischemia are least
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J. Exp. Med. 2014 Vol. 211 No. 4 595-604 595
www.jem.org/cgi/doi/10.1084/jem.20131377
understood. Several laboratories have reported reduced neuro-
logical deficit and infarct volumes at 2448 h reperfusion in
T celldeficient mice after tMCAO (Yilmaz and Granger, 2010).
After tMCAO, recombination activating gene 1deficient
(RAG1 KO) mice, which lack T and B lymphocytes, have sig-
nificantly smaller brain injury compared with controls; whereas,
adoptive transfer of WT CD4+ helper T cells or CD8+ cyto
toxic T cells increases stroke infarct volumes within 24 h after
ischemia in these mice (Kleinschnitz et al., 2010). Additionally,
TCR-transgenic mice and mice lacking co-stimulatory TCR
signaling molecules were fully susceptible to acute I/R injury,
indicating that T cell involvement at early time points is anti-
gen-independent (Kleinschnitz et al., 2010). These data dem-
onstrate that conventional CD4+ or CD8+ T cells exacerbate
acute injury after cerebral ischemia independently of TCR li-
gation, and this effect seems to be concomitant with an early
increase in T cell infiltration into the postischemic brain, which
many have reported to be between 3 and 48 h (Yilmaz et al.,
2006; Gelderblom et al., 2009).
Recent findings suggest that, in the postischemic brain,
within hours of reperfusion T cells accumulate in postcapillary
segments of periinfarct inflamed cerebral microvasculature
characterized by high endothelial expression of chemokines
and adhesion molecules. These postcapillary venules have been
postulated to allow accumulating immune cells to activate each
other and promote platelet adhesion in a process termed
thrombo-inflammation (Nieswandt et al., 2011). Much research
has been devoted to identifying T cell factors that promote
thrombo-inflammation (Barone et al., 1997; Hedtjrn et al.,
2002; Yilmaz et al., 2006; Shichita et al., 2009; Gelderblom et al.,
2012); however, to our knowledge no study has yet identified
the T cellderived factors responsible for the early increase in
infarct volumes at 24 h reperfusion. Here, we present data that
identify IL-21 as a key CD4+ T cellderived inflammatory fac-
tor that contributes to increased early ischemic tissue injury.
RESULTS AND DISCUSSION
Robust up-regulation of IL-21 during cerebral I/R injury
IL-21 is closely related to IL-2 and IL-15 and signals through
the IL-21 receptor, which is comprised of an IL-21specific
subunit and a common subunit shared with IL-2, IL-7, IL-9,
and IL-15. IL-21 is known to regulate immune responses by
promoting antibody production, T cellmediated immunity,
and NK cell and CD8+ T cell cytotoxicity. Recently, others
have shown that stress signals from necrotic tissue can induce
rapid IL-21 production from naive T cells (Holm et al., 2009),
and co-stimulation with TLR3 ligands during polyclonal T cell
activation significantly increases IL-21 secretion that contributes
to small intestine localized pediatric celiac disease (van Leeuwen
et al., 2013).To test whether IL-21 is up-regulated in brain after
ischemic necrosis induced by MCAO and to better understand
the cytokines involved in T cellmediated cerebral I/R injury,
we measured changes in inflammatory gene expression in the
brain within 24 h after tMCAO in mice using PCR-based
gene array analysis. In addition to verifying the up-regulation
596
of several previously reported inflammatory genes, we found
that IL-21 was one of the most highly expressed inflammatory
genes among those measured (Fig. 1 a). Gene expression levels
from arrays were normalized to interquartile spot intensity.
Arrays did not differ systemically in gene expression levels be-
fore or after normalization (not depicted).This increase in IL-21
gene expression was confirmed by real-time (RT) PCR analy-
sis, which detected a >24-fold relative increase in IL-21 gene
expression in the ipsilateral ischemic brain tissues compared
with the contralateral hemisphere at 24 h reperfusion (Fig. 1 b).
IL-21 was not detectable by this method in healthy brain tis-
sue (Fig. 1 b).
IL-21deficient mice are protected from acute
neuronal injury after cerebral I/R injury
Whether IL-21 contributes to ischemic tissue injury had not
been directly studied. However, in the last few years it has be-
come evident that IL-21 expression is associated with acute
rejection in mice after kidney, heart, or liver allograft (Baan et al.,
2007; Hecker et al., 2009; Xie et al., 2010). Because these
models also involve reperfusion of ischemic tissues, these find-
ings support the potential role of IL-21 in I/R injury.
We evaluated the levels of cerebral I/R injury in IL-21
deficient (IL-21 KO) mice. Infarct volumes in IL-21 KO mice
were reduced to 35% of the infarct volumes observed in con-
genic C57BL6/J WT mice as early as 24 h after tMCAO, as
measured by triphenyltetrazolium chloride (TTC; Fig. 1 c).
Similar effects were also seen at 4 d (not depicted) and 7 d after
tMCAO (Fig. 1 d), indicating that IL-21 contributes to both
immediate and delayed brain injury and suggesting that in the
absence of IL-21 tissue repair can occur. Analysis of intracra-
nial vascular anatomy revealed no differences between WT and
IL-21 KO mice in the patency of the posterior communicating
artery that would account for the observed differences in tissue
injury (Fig. 1 g). Nor did we observe differences between WT
and IL-21 KO mice in heart rate, or blood pressure before or
after tMCAO (Fig. 1 h). WT and IL-21 KO CD4+ T cells,
CD8+ T cells, and CD11b+ myeloid cells showed no difference
in IL-2, IL-17A, IFN-, or TNF production when stimulated
in vitro (Fig. 2, gl). The reduction in infarct volumes corre-
sponded with less weight loss (unpublished data), less spleen
atrophy (Fig. 2 c), and improved neurological functioning in
IL-21 KO mice compared with WT mice as assessed by both
grip strength (Fig. 1 e) and Bederson (Fig. 1 f) scoring 1, 4, and
7 d after tMCAO.
We measured accumulation of monocytes and lympho-
cytes in the brain after tMCAO in IL-21 KO and WT animals
(gating strategy in Fig. 2 a).We did not observe significant dif-
ferences in the rate of accumulation of monocytes (CD45high
CD11b+Ly6chigh), microglia (CD45intCD11b+), B cells (B220+),
T cells (TCR+), NK, or NKT cells (NK1.1+) in the
ischemic brain of WT and IL-21 KO mice after 1, 4, and 7 d
of reperfusion (unpublished data). In contrast, as early as 1 d
after tMCAO, IL-21 KO mice showed significantly dimin-
ished cerebral accumulation of CD4+ T cells and CD8+ T cells
IL-21 promotes brain injury after stroke | Clarkson et al.

after tMCAO compared with WT mice (Fig. 2 e) and these
differences persisted at day 7 (Fig. 2 f ). These differences were
not reflected in the spleen before or after tMCAO (Fig. 2 b).
IL-21 has also been shown to be produced by and modulate
the function of regulatory T cells (Peluso et al., 2007; Battaglia
et al., 2013), which begin to accumulate in the brain after
tMCAO. Thus, we compared the frequency of regulatory
CD4 T cells expressing the marker Foxp3 in the brain and
spleen 24 h after tMCAO in WT and IL-21 KO mice. IL-21
KO mice exhibited no difference in regulatory T cell abundance
JEM Vol. 211, No. 4
Br ief Definitive Repor t
Figure 1. IL-21 is up-regulated early in
mouse brain, and IL-21deficient mice are
protected after tMCAO. GeArray S Series
Mouse Autoimmune and Inflammatory Re-
sponse gene array of transcripts expressed in
pooled brain tissues 24 h after tMCAO or
sham procedure (n = 36 mice per group).
(a) Bar graphs show PCR array spot intensity
of genes with a greater than sixfold difference
in gene expression in tMCAO compared with
sham, normalized to the interquartile mean
spot intensity. (b) IL-21 mRNA expression
level in ipsilateral hemisphere relative to con-
tralateral hemisphere 24 h after tMCAO (n = 3
per group). Mann-Whitney rank sum test
*, P < 0.05. Plots show median, lower, and upper
quartile (box) and range (error bars). Infarct
volumes of WT and IL-21 KO mice 24 h after
tMCAO (c) and 7 d after tMCAO (d). Represen-
tative images of TTC-stained 2-mm brain
slices shown below (n = 57 mice per group).
WT and IL-21 KO mice grip strength (e) and
Bederson score (f) at 1, 4, and 7 d after 1 h
tMCAO (n = 78 for each group). (g) Brain
vasculature of C57BL/6 and IL-21KO mice
perfused transcardially with carbon lampblack
(C198-500; Thermo Fisher Scientific) in 20%
gelatin ddH2O. Arrows indicate anterior cere-
bral (ACA), MCA, posterior cerebral (PCA),
basilar (BA), and vertebral arteries (VA), show-
ing point of occlusion (white circle). High
magnification images demonstrate no differ-
ence in patency of the posterior communicat-
ing artery (PComA, arrows). (h) Heart rate and
blood pressure of WT and IL-21 KO mice be-
fore and after tMCAO (n = 34 mice per
group). Data are representative of 23 inde-
pendent experiments. **, P < 0.01; ***, P <
0.001; ****, P < 0.0001 by Students t test
(single comparison) or one-way ANOVA (mul-
tiple comparisons). Error bars indicate SD.
in either tissue compared with WT experimental animals. Nor
did we observe a difference between WT and IL-21 KO mice
in the frequency of lymphocytes producing the antiinflam-
matory cytokine IL-10 among B cells (B220+), CD8+ T cells,
or CD4+ T cells (unpublished data). These data demonstrate
that IL-21 deficiency is protective at acute time points after
tMCAO and IL-21 levels in the CNS correlate with early in-
filtration of T cells without affecting regulatory T or B cell
accumulation or IL-10 cytokine production during the acute
period (day 14).
597
Figure 2. Lymphocyte recruitment to brain is diminished in IL-21 deficient mice. (a) Gating strategy for leukocytes isolated from brain after
MCAO. (b) WT and IL-21 KO spleen cells 24 h after tMCAO or sham procedure (n = 3 mice per group). (c) Relative change in spleen weight of WT and IL-21
KO mice after tMCAO (n = 37 mice per group). (d) Percentage of blood and spleen CD4+ T cells expressing IL-21 after 5-h ex vivo stimulation with PMA
(10 ng/ml) and Ionomycin (1 g/ml) 4 d after MCAO or control treatment. (e and f) Leukocyte accumulation in the brain of WT mice compared with IL-21
KO mice 1, 4, and 7 d after tMCAO (n = 36 mice per group). (gk) In vitro cytokine expression by WT and IL-21 KO CD4+ and CD8+ T cells after 5-h
stimulation under indicated conditions with or without recombinant mouse IL-21 (100 ng/ml). (l) TNF production by CD11b+ myeloid cells stimulated with
LPS (500 ng/ml) for 5 h with or without recombinant mouse IL-21 (100 ng/ml). Cells isolated from n = 3 mice per group. Data are representative of two to
four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Students t test (single comparison) or one-way ANOVA (multiple
comparisons). Error bars indicate SD (bd and gl) and SEM (e, f).

598 IL-21 promotes brain injury after stroke | Clarkson et al.

 Br ief Definitive Repor t

IL-21 is primarily produced IL-21 blockade is protective in tMCAO

by brain-infiltrating CD4+ T cells
We measured intracellular IL-21 production by various cell
populations. IL-21producing cells were not detected by flow
cytometry among cells isolated from healthy brain, but could
be detected among mononuclear cells isolated from isch-
emic brain 24 h after tMCAO. Gating on IL-21+ cells revealed
that the majority of these cells were CD4+ T cells (Fig. 3 a).
IL-21producing CD4+ T cells were also detected at low lev-
els among cells isolated from blood, but not spleen, of healthy
WT mice, and these levels were unaffected after transient ce-
rebral ischemia (Fig. 2 d), indicating that the increase in IL-21
production was limited to CD4+ T cells recovered from the
postischemic brain. Next, we adoptively transferred WT or
IL-21 KO CD4+ T cells into lymphocyte-deficient RAG2
KO mice. Purity of transferred CD4+ T cells was confirmed
by flow cytometry to be 95% (Fig. 3 b). As shown previ-
ously (Kleinschnitz et al., 2010), we observed markedly re-
duced infarcts in RAG KO mice compared with WT mice,
and infarct volumes could be restored to WT levels in RAG2
KO by adoptively transferring WT CD4+ T cells. Most im-
portantly, RAG2 KO mice that received WT CD4+ T cells
had significantly larger infarcts than those receiving IL-21
KO CD4+ T cells (Fig. 3 c).
We treated WT mice with IL-21 receptor Fc protein (IL-21R.
Fc) using a previously described protocol (Jang et al., 2009;
McGuire et al., 2011; Spolski et al., 2012). We administered
500 g of IL-21R.Fc i.p. 1 h before tMCAO. As measured by
TTC staining, treated mice showed significantly reduced in-
farct volumes compared with control-treated mice 24 h after
tMCAO (Fig. 4 a). We found a similarly protective effect in
mice treated with IL-21R.Fc protein (500 g i.p.) 2 h after ini-
tiation of reperfusion (Fig. 4 a). These differences were associ-
ated with decreased locomotor function (decreased resistance
to lateral push and increased circling behavior) in control-
treated mice compared with those treated with IL-21R.Fc
(Fig. 4 b and Video 1). Although we cannot exclude the pos-
sibility that IL-21R.Fc exhibits its blocking effect in periph-
eral immune compartments, using ELISA for human IgG4 we
were able to observe thatupon i.p. injectionsoluble IL-21R.
Fc accumulates in the CNS of mice after tMCAO (Fig. 4 c).
IL-21 presence localizes with CD4 staining
in human stroke tissue
IL-21+ cells were recently detected in human brain tissue
during different neuroinflammatory conditions (Tzartos et al.,
2011).Thus, we stained groups of postmortem brain tissue from

Figure 3. IL-21 is primarily produced by brain-infiltrating CD4+ T cells. (a) Intracellular cytokine staining of lymphocytes isolated from n = 5
pooled healthy WT, ischemic IL-21/, or ischemic WT mouse brains 24 h after tMCAO or sham procedure showing IL-21 versus CD8 expression. Histo-
grams show CD4, NK1.1, and TCR expression on IL-21+ cells from ischemic WT brain. (b) CD45, CD4, and LFA-1 expression by negative fractions purified
from WT and IL-21/ lymph node cells by CD4+ negative selection using magnetic cell separation before transfer into RAG2/ recipients. (c) Infarct
volume in WT mice (n = 4), RAG2/ mice (n = 4), RAG2/ mice + WT CD4 T cells (n = 10), and RAG2/ mice + IL-21/ CD4 T cells (n = 10) 24 h
after tMCAO. Representative TTC-stained 2-mm mouse brain slices shown on top. Data are representative of two independent experiments. **, P < 0.01;
***, P < 0.001; ****, P < 0.0001 one-way ANOVA. Error bars indicate SD.

JEM Vol. 211, No. 4 599

Figure 4. Blockade of IL-21 signaling before or after tMCAO reduces infarct size in WT mice. (a) Infarct volumes 24 h after tMCAO in WT mice
treated with 500 g recombinant mIL-21R.Fc or PBS 1 h before (pretreatment) or 2 h after (posttreatment) surgery. Representative TTC-stained brain
slices shown on left (n = 34 mice per group). (b) Still image from Video 1 depicting behavioral differences between WT mice posttreated with IL-21R.Fc
or PBS. (c) IL-21R.Fc protein levels in the indicated organs 2024 h after tMCAO in WT mice injected with 500 g IL-21R.Fc 2 h after start of reperfusion
(n = 24 mice per group). N.D., not detected. Data are representative of two independent experiments. **, P < 0.01; ***, P < 0.001, by Students t test. Error
bars indicate SEM. Representative images of postmortem paraffin-embedded human acute stroke lesions stained with control sera (d), or primary anti-
bodies against CD4 (e and g [ii-iii]), IL-21 (f and g [iii]), or eosin (g [i]) visualized with Fast Red (d, e, and g) and/or DAB (d, f, and g [iii]) and counterstained
with hematoxylin. High magnification images are shown on right. Arrows indicate positive staining. Bars, 50 m.

600 IL-21 promotes brain injury after stroke | Clarkson et al.


patients with acute and chronic stroke lesions. In acute infarcts,
rare CD4+ T cells were found in the necrotic brain parenchyma
(Fig. 4 g, ii, arrows), which was predominantly infiltrated by
foamy macrophages (Fig. 4 g, i). In contrast, CD4+ T cells were
consistently found within the Virchow Robins space of vessels
bordering acute infarcts (not depicted) and in the subarachnoid
space adjacent to meningeal vessels (arrows, Fig. 4 e, i). IL-21
staining was limited almost exclusively to these perivascular
spaces. Compared with control stained tissue (Fig. 4 d, i), anti
IL-21 staining labeled cells extensively in the subarachnoid
space of deep sulci penetrating the infarcted tissueshowing
a similar distribution to CD4+ cells in serial sections (arrows,
Fig. 4 f ). Additionally, double staining with antibodies for
CD4 and IL-21 revealed the presence of CD4+ IL-21+ cells
in this perivascular niche (arrow, Fig. 4 g, iii). In summary, CD4+
JEM Vol. 211, No. 4
Br ief Definitive Repor t
T cells that can secrete IL-21 were detected within the
CSF-filled subarachnoid and perivascular spaces during cere-
bral infarction in humans.
Neuronal cells express IL-21 receptor and up-regulate
autophagy genes in response to IL-21
RT-PCR analysis of primary mouse neurons and murine neu-
ronal cell lines (Neuro2A) indicated that IL-21R expression
was higher on neuronal cells than on other brain cells, includ-
ing astrocytes and endothelial cells (Fig. 5, a and c). This is con-
sistent with another report where in situ hybridization detected
neuron-restricted IL-21R expression in inflamed human brain
tissue (Tzartos et al., 2011). Moreover, treating Neuro2a cells
with IL-21 after in vitro oxygen glucose deprivation (OGD)
significantly increased cell death as measured by XTT cell
Figure 5. IL-21 promotes autophagy expres-
sion in neuronal cells after hypoxia/ischemia.
(a) Il21r mRNA expression relative to GAPDH
expression levels is shown in normoxic and hy-
poxic primary mouse neurons after OGD or con-
trol treatment. (b) Viability of Neuro2A cells
treated with the indicated doses of IL-21 after
OGD. (c) Il21r mRNA expression relative to
GAPDH in neuronal (Neura2A), astrocytic, and
endothelial cell lines (MB114) expressed relative
to BMDC expression. (d) ATG6 expression in pri-
mary neurons treated with PBS, etoposide, or
32256 ng/ml rIL-21 for 4 h after 12 h oxygen
glucose deprivation as measured by RT-PCR. Cells
treated in triplicate. (e) Number of ATG6+ cells per
field in the same regions of WT and IL-21 KO
mouse brains after tMCAO as assessed by im-
mune staining (n = 3 mice per group). Arrows
indicate ATG-6+ cells in periinfarcted brain tissue
of WT and IL-21 KO mice. Bars, 100 m. Data
are representative of two independent experi-
ments. *, P < 0.05; **, P < 0.01; ***, P < 0.001;
****, P < 0.0001 by Students t test (single com-
parison) or one-way ANOVA (multiple compari-
sons). Error bars indicate SEM.
601
viability assay (Fig. 5 b). In subsequent studies we found that
treatment of primary neurons with IL-21 up-regulated mRNA
levels of the autophagy associated gene ATG6 (Fig. 5 d). These
data suggest that IL-21 could directly affect neuronal autoph-
agy during ischemic injury, which has been implicated in neu-
ronal death in infarcted and periinfarcted brain tissue.Thus, we
stained WT and IL-21 KO postischemic brain tissues for ATG6.
We observed significantly fewer ATG6+ cells in infarcted brain
tissue of IL-21 KO mice compared with WT, suggesting that
IL-21 may contribute to increased cerebral autophagy after
stroke (Fig. 5 e).
In conclusion, we implicate IL-21 as a lymphocyte-derived
factor with a pronounced effect on brain injury after focal isch-
emia in mice. We also present data demonstrating that IL-21
producing CD4+ T cells are present in the brain of patients
with acute stroke.These data warrant investigation of the thera-
peutic potential of IL-21modifying treatments in isolation
and combination with current anti-thrombotic treatments for
ischemic stroke.
MATERIALS AND METHODS
Ethics statement. C57BL/6 WT mice were obtained from The Jackson
Laboratory. IL-21deficient mice (IL-21tm1Lex) were purchased from the Mu-
tant Mouse Regional Resources Center. All mice underwent 1 h tMCAO
and 24 h reperfusion. All animal procedures used in this study were con-
ducted in strict compliance with the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and approved by the University of
Wisconsin Center for Health Sciences Research Animal Care Committee.
All mice (25 g) were anesthetized with 5% halothane for induction and
1.0% halothane for maintenance vaporized in N2O and O2 (3:2), and all ef-
forts were made to minimize suffering.
Regional cerebral blood flow (rCBF) measurement. Changes in rCBF
at the surface of the left cortex were recorded using a blood perfusion monitor
(Laserflo BPM2;Vasamedics) with a fiber optic probe (0.7 mm diam). The tip
of the probe was fixed with glue on the skull over the core area supplied by the
MCA (2 mm posterior and 6 mm lateral from bregma). Changes in rCBF after
MCAO were recorded as a percentage of the baseline value. Mice included in
these investigations had >80% relative decrease in rCBF during MCAO.
Investigation of intracranial vasculature. WT and IL-21 KO mice were
anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.).
After thoracotomy was performed, a cannula was introduced into the ascend-
ing aorta through the left ventricle. Transcardial perfusion fixation was per-
formed with 2 ml saline and 2 ml of 3.7% formaldehyde. Carbon lampblack
(C198-500; Thermo Fisher Scientific) in an equal volume of 20% gelatin in
ddH2O (1 ml) was injected through the cannula. The brains were removed
and fixed in 4% PFA overnight at 4C. Posterior communicating arteries
(PComA) connect vertebrobasilar arterial system to the Circle of Willis and
internal carotid arteries, and its development affects brain sensitivity to isch-
emia among different mouse strains (Barone et al., 1993). Development of
PComA in both hemispheres was examined and graded on a scale of 03, as
reported previously (Majid et al., 2000). 0, no connection between anterior
and posterior circulation; 1, anastomosis in capillary phase (present but poorly
developed); 2, small truncal PComA; 3, truncal PComA.
Focal ischemia model. Focal cerebral ischemia in mice was induced by oc-
clusion of the left MCA, as described previously (Longa et al., 1989). Operators
performing surgeries were masked to experimental groups. In brief, the left
common carotid artery was exposed, and the occipital artery branches of the
external carotid artery (ECA) were isolated and coagulated. After coagulation
602
of the superior thyroid artery, the ECA was dissected distally and coagulated
along with the terminal lingual and maxillary artery branches.The internal ca-
rotid artery (ICA) was isolated, and the extracranial branch of the ICA was then
dissected and ligated. A standardized polyamide resin glue-coated 6.0 nylon
monofilament (3021910; Doccol Corp) was introduced into the ECA lumen,
and then advanced 99.5 mm in the ICA lumen to block MCA blood flow.
During the entire procedure, mouse body temperature was kept between 37
and 38C with a heating pad. The suture was withdrawn 60 min after occlu-
sion. The incision was closed, and the mice underwent recovery.
Infarction size measurement. After 24 h reperfusion, mice were sacrificed
and brains were removed and frozen at 80C for 5 min. 2-mm coronal
slices were made with a rodent brain matrix (Ted Pella, Inc.). The sections
were stained for 20 min at 37C with 2% TTC (Sigma-Aldrich). Infarction
volume was calculated with the method reported by Swanson et al. (1990) to
compensate for brain swelling in the ischemic hemisphere. In brief, the sec-
tions were scanned, and the infarction area in each section was calculated by
subtracting the noninfarct area of the ipsilateral side from the area of the
contralateral side with National Institutes of Health image analysis software,
ImageJ. Infarction areas on each section were summed and multiplied by sec-
tion thickness to give the total infarction volume.
Gene array and RT-PCR. Ipsilateral brain hemispheres were dissected and
stored in RNAlater (QIAGEN) at 4C until further use. Total RNA was ex-
tracted and purified with RNeasy Protect Mini kit (QIAGEN) according to
the manufacturers instructions. Purified RNA samples were analyzed by
GeArray S Serious Mouse Autoimmune and Inflammatory Gene array
(SuperArray; Bioscience Corporation). Results from GeArray were filtered
for genes with spot intensities higher than the mean local background of the
bottom 75% of nonbleeding spots. For RT-PCR, 1 g total RNA from each
sample was reverse transcribed using SuperScript II first strand cDNA syn-
thesis kit (Invitrogen). RT-PCR was performed on a Smart Cycler (Model SC
1001; Cepheid) using IL-21 TaqMan gene expression assay (Mm00517640_m1;
Applied Biosystems), RT2 qPCR Primer assay for mouse Becn1
(PPM32434A; SABiosciences), or RT2 qPCR Primer Assay for Mouse IL21r
(PPM03762A; SABiosciences). The data were normalized to an internal ref-
erence gene, GAPDH.
Mononuclear cell isolation and flow cytometry. Brains were removed
from perfused animals, weighed, minced, transferred to Medicon inserts, and
ground in a MediMachine (BD) for 2030 s.The cell suspension was washed
with HBSS, and cells were resuspended in 70% Percoll (Pharmacia) and over-
laid with 30% Percoll. The gradient was centrifuged at 2,250 g for 30 min at
4C without brake. The interface was removed and washed once for further
analysis. CD11b-positive and -negative fractions were isolated using Imag
anti-CD11b magnetic particles (BD), following the manufacturers protocol.
A total of 106 cells were incubated for 30 min on ice with saturating concen-
trations of labeled antibodies with 40 g/ml unlabeled 2.4G2 mAb to block
binding to Fc receptors, and then washed 3 times with 1% BSA in PBS.
Single-cell suspensions from various tissues were cultured at 37C in
10% FBS in RPMI 1640 media supplemented with GolgiStop (BD) in the pres-
ence of either phorbol myristate acetate (50 ng/ml) and ionomycin (1 g/ml)
for 5 h. After surface staining with antibodies against CD4, NK1.1, and
TCR, cell suspensions were fixed and permeabilized by Cytofix/Cy-
toperm solution (BD), followed by staining with antiIL-21 antibodies.
Fluorochrome-labeled antibodies against CD45, CD11b, Ly6c, B220, CD4,
CD8a, NK1.1, IFN-, and appropriate isotype controls were purchased from
BD. Fluorochrome-labeled antibody against IL-21 and TCR was pur-
chased from eBioscience. Cell staining was acquired on a FACSCalibur or
LSRII (BD) and analyzed with FlowJo (Tree Star) software version 5.4.5.
Neurofunctional assessment. Neuromuscular coordination was assessed by
grip strength test, as previously described (Kleinschnitz et al., 2010). For this
test, mice were placed on a horizontal string midway between two supports.
Mice were scored from 0 to 5 as follows: 0, falls off within 2 s; 1, hangs on with
IL-21 promotes brain injury after stroke | Clarkson et al.

forepaw(s); 2, hangs on with forepaws and moves laterally on string; 3, hangs
onto string with forepaws and hindpaw(s); 4, hangs onto string with forepaws,
hindpaw(s) and tail; 5, escape to supports. Mice were allowed to rest between
trials. Scores for each mouse were determined by averaging 510 trials (each
lasting 15 s). Global neurological deficit was determined by a modified Bed-
erson scoring system: 0, no deficit; 1, forelimb flexion; 2, unidirectional circling
after being lifted by tail; 3, spontaneous unidirectional circling; 4, longitudinal
rolling upon being lifted by tail; 5, spontaneous longitudinal rolling.
Generation of IL-21 receptor Fc fusion protein. Chinese hamster ovary
cell line (Korn et al., 2007) expressing the extracellular domain (aa 20236)
of mouse IL-21R fused to the fragment crystallizable (Fc) portion of human
IgG4 (IL-21R.Fc) were maintained in UltraCHO (BioWhittaker). IL-21R.
Ig was purified from the culture supernatant by passage through a protein
GSepharose column and concentrated by ultrafiltration. Concentration was
determined spectrophotometrically. Purity and molecular weight were con-
firmed by sodium dodecyl-sulfate PAGE and human-IgG4 ELISA (eBiosci-
ence) following the manufacturers instructions. The IL-21R.Fc reagent was
tested in vitro for its ability to suppress IL-21induced T cell proliferation.
Immunohistochemistry. Paraffin-embedded postmortem brain tissue sec-
tions from individuals with acute and chronic stroke lesions were obtained
from the Neuropathology Laboratory of the University of Wisconsin De-
partment of Pathology. After rehydration and deparaffinization, sections un-
derwent heat-induced antigen retrieval in 10 mM sodium citrate, pH 6.0, for
surface antigens or Tris-EDTA (10 mM/1 mM) with 0.05% Tween-20 for
intracellular antigens. Sections were blocked for 30 min with secondary
serum (10% in Tris-buffered saline) and then stained with primary antibodies,
0.5% chicken antiIL-21 (Lifespan Biosciences) or prediluted mouse anti-
CD4 ([1F6]; ab17131; Abcam) for 12 h at 37C or overnight at 4C. Nor-
mal primary sera (510%) were used for negative control. After several washes,
secondary antibodies (biotin-labeled goat antichicken or biotin-labeled
goat antimouse;Vector Laboratories) were applied to sections and incubated
for 2 h at room temperature. Staining was developed using the VECTA-
STAIN ABC-HRP kit (Vector Laboratories) with diaminobenzidine sub-
strate (BD) or streptavidin-alkaline phosphatase with Fast Red substrate
(Laboratory Vision), following the manufacturers instructions. Slides were
lightly counterstained with hematoxylin, rinsed with running tap water, and
mounted. For frozen mouse sections WT and IL-21 KO mice underwent 1-h
tMCAO and 17-d reperfusion. Brains were perfused with PBS and 3% for-
malin, embedded in OCT, and cut into 8-m frozen sections for immuno
histochemistry. Frozen sections were thawed for 10 min at room temperature
and blocked with 5% goat serum solution in PBS for 15 min. Sections were
stained with rabbit polyclonal antibodies for ATG6 for 1 h at room tempera-
ture, followed by phycoerythrin-labeled goat antirabbit IgG (Santa Cruz
Biotechnology, Inc.). Images were acquired on a BX40 microscope equipped
with a Q-Color 3 camera using Q-Capture software (Olympus). Digital
images were processed and analyzed using Photoshop CS4 software (Adobe).
Color balance, brightness, and contrast settings were manipulated to generate
final images. All changes were applied equally to entire image.
Neuronal cell oxygen glucose deprivation. Primary neuronal cultures
derived from embryonic day 1418 mouse cortices were grown to 80% con-
fluency in neural basal media supplemented with B27 (2%) and penicillin/
streptomycin (1%), as previously described (Kintner et al., 2010). Astrocytic
and microglial contamination was excluded based on the absence of GFAP+
and CD11b+ cells when stained by immunocytochemistry. For OGD, media
was replaced with neural basal media with or without glucose and placed in
a hypoxic chamber or under normoxic conditions for 2 h at 37C. Afterward,
cells were lysed and mRNA isolated using RNeasy mini kit (QIAGEN). For
XTT viability assay, Neuro2A underwent OGD and were treated with dose
curve of IL-21 immediately after return to normoxic media and incubated
for 4 h at 37C. XTT labeling mixture (50 l per well; Roche) was added ac-
cording to manufacturers instructions, and at18 h fluorescence was read on
a GENious Microplate reader (Tecan). Cell number was calculated using a
standard curve of known untreated cells kept under normoxic conditions.
JEM Vol. 211, No. 4
Br ief Definitive Repor t
Statistical analyses and quality standards. All surgeries were performed
in a blinded manner by a third party and measurements masked where possi-
ble. Infarct volume measurements from TTC stained sections were averaged
from two to three independent blinded observers. Based on power calcula-
tions, n = 310 sex- and age-matched mice were used for each experiment
and group assignment was randomized. Among animals receiving MCAO
procedure, 86.5% of WT mice, 93.5% of IL-21KO mice, and 100% of
RAG2KO mice were included in analysis. Mice were excluded due to prema-
ture death (13.5% of WT mice, 3.2% of IL-21 KO mice) or vessel variation
(3.2% of IL-21KO mice). Results are given as means 1 SD. Multiple com-
parisons were made using one-way ANOVA. Where appropriate, two-tailed
Students t test analysis was used for comparing measures made between two
groups. For comparison of RT-PCR data, nonparametric Mann-Whitney
rank sum analysis was used. P-values <0.05 were considered significant.
Online supplemental material. Video 1 shows groups of WT C57BL6
mice treated with 500 g IL-21R.Fc or PBS control via i.p. injection. On-
line supplemental material is available at http://www.jem.org/cgi/content/
full/jem.20131377/DC1.
We thank Satoshi Kinoshita for expert histopathology services, Guoqing Song for
assisting in the surgical procedures, Dr. Wenda Gao for providing reagents and
protocols for the purification of IL-21R.Fc protein, and members of our laboratory
for helpful discussions and constructive criticisms of this work. We also thank Khen
Macvilay and Sinarack Macvilay for their expertise provided for cytofluorimetry and
immunohistochemistry studies and Samuel (Joe) Ollar for assisting in the OGD procedure.
This work was supported by awards from the American Heart Association (pre-
doctoral fellowship #12PRE12060020 to B.D.S. Clarkson) and the National Institutes
of Health (NS037570 and NS076946 to ZF, AI048087 to M.S. Salamat, and
AI068730 to J.D. Lambris).
The authors have no competing financial interests.
Submitted: 1 July 2013
Accepted: 24 February 2014
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