Inhibiting CXCR3-Dependent CD8+ T Cell Trafficking Enhances Tolerance Induction in a Mouse Model of Lung Rejection
ABSTRACT Lung transplantation remains the only effective therapy for patients with end-stage pulmonary diseases. Unfortunately, acute rejection of the lung remains a frequent complication and is an important cause of morbidity and mortality. The induction of transplant tolerance is thought to be dependent, in part, on the balance between allograft effector mechanisms mediated by effector T lymphocytes (Teff), and regulatory mechanisms mediated by FOXP3(+) regulatory T cells (Treg). In this study, we explored an approach to tip the balance in favor of regulatory mechanisms by modulating chemokine activity. We demonstrate in an adoptive transfer model of lung rejection that CXCR3-deficient CD8(+) Teff have impaired migration into the lungs compared with wild-type Teff, which results in a dramatic reduction in fatal pulmonary inflammation. The lungs of surviving mice contained tolerized CXCR3-deficient Teff, as well as a large increase in Treg. We confirmed that Treg were needed for tolerance and that their ability to induce tolerance was dependent on their numbers in the lung relative to the numbers of Teff. These data suggest that transplantation tolerance can be achieved by reducing the recruitment of some, but not necessarily all, CD8(+) Teff into the target organ and suggest a novel approach to achieve transplant tolerance.
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ABSTRACT: BACKGROUND: Induction of endogenous regulatory T (Treg) cells represents an exciting new potential modality for treating allergic diseases, such as asthma. Treg cells have been implicated in the regulation of asthma, but the anatomic location in which they exert their regulatory function and the mechanisms controlling the migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg cell biology will be important for harnessing their power in the clinic. OBJECTIVE: We sought to determine the anatomic location at which Treg cells exert their regulatory function in the sensitization and effector phases of allergic asthma and to determine the chemokine receptors that control the migration of Treg cells to these sites in vivo in both mice and human subjects. METHODS: The clinical efficacy and anatomic location of adoptively transferred chemokine receptor-deficient CD4(+)CD25(+) forkhead box protein 3-positive Treg cells was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Treg cells recruited into the human airway after bronchoscopic segmental allergen challenge of asthmatic patients. RESULTS: We show that CCR7, but not CCR4, is required on Treg cells to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Treg cells to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, human subjects with allergic asthma had an increase in CCR4-expressing functional Treg cells in the lungs after segmental allergen challenge. CONCLUSION: The location of Treg cell function differs during allergic sensitization and allergen-induced recall responses in the lung, and this differential localization is critically dependent on differential chemokine function.The Journal of allergy and clinical immunology 04/2013; · 12.05 Impact Factor
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ABSTRACT: Objective: Chemokine (C-X-C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regulation of immune cell trafficking and activation. We observed increased CXCR3 expression in the visceral adipose of obese humans and mice. We hypothesized a pathophysiologic role for CXCR3 in diet-induced obesity (DIO). Design and Methods: Wild type C57B/L6J (WT) and chemokine receptor 3 knockout (CXCR3(-/-) ) mice were fed a high fat diet (HFD) for 20 weeks followed by assessment of glucose metabolism and VAT inflammation. Results: CXCR3(-/-) mice exhibited lower fasting glucose and improved glucose tolerance compared to WT-HFD mice, despite similar body mass. HFD-induced VAT innate and adaptive immune cell infiltration, including immature myeloid cells (CD11b(+) F4/80(lo) Ly6C(+) ), was markedly ameliorated in CXCR3(-/-) mice. In vitro IBIDI and in vivo migration assays demonstrated no CXCR3-mediated effect on macrophage or monocyte migration, respectively. CXCR3(-/-) macrophages, however, had a blunted response to interferon-γ, a TH 1 cytokine that induces macrophage activation. Conclusions: We demonstrate a previously unreported role for CXCR3 in the development of HFD-induced insulin resistance and VAT macrophage infiltration in mice. Our results support pharmaceutical targeting of the CXCR3 receptor as a potential treatment for obesity/insulin resistance.Obesity 10/2013; · 3.92 Impact Factor
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ABSTRACT: Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.PLoS ONE 01/2013; 8(8):e72955. · 3.73 Impact Factor
The Journal of Immunology
Inhibiting CXCR3-Dependent CD8+T Cell Trafficking
Enhances Tolerance Induction in a Mouse Model of Lung
Edward Seung,*,†Josalyn L. Cho,*,‡Tim Sparwasser,xBenjamin D. Medoff,*,‡
and Andrew D. Luster*,†
Lung transplantation remains the only effective therapy for patients with end-stage pulmonary diseases. Unfortunately, acute re-
jection of the lung remains a frequent complication and is an important cause of morbidity and mortality. The induction of trans-
plant tolerance is thought to be dependent, in part, on the balance between allograft effector mechanisms mediated by effector
T lymphocytes (Teff), and regulatory mechanisms mediated by FOXP3+regulatory T cells (Treg). In this study, we explored
an approach to tip the balance in favor of regulatory mechanisms by modulating chemokine activity. We demonstrate in an
adoptive transfer model of lung rejection that CXCR3-deficient CD8+Teff have impaired migration into the lungs compared with
wild-type Teff, which results in a dramatic reduction in fatal pulmonary inflammation. The lungs of surviving mice contained
tolerized CXCR3-deficient Teff, as well as a large increase in Treg. We confirmed that Treg were needed for tolerance and that
their ability to induce tolerance was dependent on their numbers in the lung relative to the numbers of Teff. These data suggest
that transplantation tolerance can be achieved by reducing the recruitment of some, but not necessarily all, CD8+Teff into the
target organ and suggest a novel approach to achieve transplant tolerance.
therapies and surgical techniques, overall median survival after
lung transplantation is only 5 y, with actuarial survival at 77, 61,
and 50% at 1, 3, and 5 y, respectively (3). Clinical studies have
implicated lung injury from acute rejection (AR) as a major
causative factor of morbidity and mortality after lung trans-
The holy grail of the transplantation field is to induce donor-
specific tolerance. In animal models, long-term graft survival and
transplantation tolerance without immunosuppression can be in-
duced by a number of methods (7). Most of these strategies have
relied on the concept that deletion or inhibition of donor-specific
effector T lymphocytes (Teff) is necessary to prevent rejection and
achieve tolerance (8, 9). Recently, it has become clear that regu-
The Journal of Immunology, 2011, 186: 6830–6838.
ung transplantation remains the only effective therapy for
the large number of patients with end-stage lung disease
(1, 2). However, despite advances in immunosuppressive
latory T cells (Treg) play a crucial role in suppressing alloimmune
responses directed against transplanted tissues (10). Naturally
occurring and induced Treg have been identified as CD4+CD25+
T lymphocytes that specifically express the forkhead family
transcription factor FOXP3 (11–13) and are critical regulators of
autoimmunity (14) and peripheral tolerance (15, 16). A large body
of data has emerged suggesting tolerance depends on a balance
between effector mechanisms mediated by Teff and suppressive
mechanisms mediated by Treg (17). In the presence of low ef-
fector cell numbers, regulatory mechanisms are thought to sup-
press effector mechanisms, keeping them in check, resulting in
transplant tolerance. Unfortunately, many of the immunosup-
pressive techniques used to prevent rejection also inhibit Treg,
thus preventing the induction of tolerance (18, 19).
Central to the development of AR is the recruitment of Teff into
the transplanted lung (20–22). Leukocyte recruitment into tissue
is orchestrated by chemoattractants, such as chemokines, a super-
family of secreted chemotactic cytokines, as well as lipid medi-
ators, which regulate cell migration through G protein-coupled
chemoattractant receptors expressed on immune cells (23–26). AR
of an organ is a complex and intense inflammatory response with
many chemokines and their receptors implicated in the process.
Research in humans and animals has demonstrated that the che-
mokine receptor CXCR3 and its ligands, CXCL9 and CXCL10,
play important roles in this process (27–29). In animal models of
heart and small bowel transplantation, the inhibition or deletion
of CXCL9, CXCL10, and CXCR3 significantly prolonged graft
survival (30–33), which conceptualized their importance in organ
rejection. Recent findings using tracheal transplantation in mice as
a model of lung transplantation have shown similar importance for
CXCR3 and its ligands (34). However, other studies have ques-
tioned the importance of CXCR3 in heart transplantation (35, 36).
CXCR3 is expressed on multiple cell types in addition to Teff,
such as NK and NKT cells, dendritic cells, B cells, and Treg (29,
37–39). The prior studies described above did not examine in-
hibition of CXCR3 on individual cell types, which may explain
*Center for Immunology and Inflammatory Diseases, Massachusetts General Hospi-
tal and Harvard Medical School, Charlestown, MA 02129;†Division of Rheumatol-
ogy, Allergy and Immunology, Massachusetts General Hospital and Harvard Medical
School, Charlestown, MA 02129;‡Pulmonary and Critical Care Unit, Massachusetts
General Hospital and Harvard Medical School, Charlestown, MA 02129; andxInsti-
tute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical
Infection Research, 30625 Hannover, Germany
Received for publication April 1, 2010. Accepted for publication April 6, 2011.
This work was supported by the Roche Organ Transplantation Research Foundation,
by National Institutes of Health Grant R01CA069212 (to A.D.L.), and by the In-
ternational Society for Heart and Lung Transplantation (to E.S.).
Address correspondence and reprint requests to Dr. Andrew D. Luster or Dr. Benja-
min D. Medoff, Massachusetts General Hospital-East, 149 13th Street, Charlestown,
MA 02129 (A.D.L.) or Massachusetts General Hospital, 55 Fruit Street, Bulfinch
148, Boston, MA 02114 (B.D.M.). E-mail addresses: email@example.com
(A.D.L.) and firstname.lastname@example.org (B.D.M.)
The online version of this article contains supplemental material.
Abbreviations used in this article: AR, acute rejection; BAL, bronchoalveolar lavage;
DT, diphtheria toxin; Teff, effector T lymphocytes; Treg, regulatory T cells; WT,
some of the variability in the literature. Using our recently de-
veloped adoptive transfer mouse model of lung rejection (21), in
the current study we address this possible confounding issue by
having CD8+T cells be the only cell type deficient in CXCR3.
In our previous study, we were able to partially inhibit Teff re-
cruitment into the lung during AR and prolong allograft survival by
specifically deleting the leukotriene B4receptor BLT1 only on Teff
(21, 34). In the current study, we evaluated the ability of CXCR3
to mediate Teff recruitment in our model of acute lung rejection,
which has advantages over established murine models by utilizing
the whole lung and having survival as an end point (21). In ad-
dition, our adoptive transfer transgenic mouse model allowed us to
specifically isolate a role for CXCR3 in the trafficking of Ag-
specific Teff. We found that deleting CXCR3 on Teff also par-
tially reduced Teff homing into the lung, and this was sufficient to
induce tolerance and prevent rejection despite recruitment of some
Ag-specific Teff into the lung. This occurred without inhibiting
T cell activation and without general immunosuppression. In ad-
dition, we observed a large increase in endogenous Treg specifi-
cally in the lungs of these mice, and these cells were essential in
inducing tolerance as Treg-deficient mice were not able to tolerize
Teff and did not survive. Taken together, these studies suggest the
novel concept that manipulation of chemoattractant-induced T cell
recruitment into the lung can generate a microenvironment ad-
vantageous to endogenous Treg. In fact, even partial inhibition of
Teff homing into the lung can tip the balance in favor of Treg and
allograft tolerance induction, thus providing a novel therapeutic
approach to solid organ transplantation.
Materials and Methods
Wild-type (WT) C57BL/6 mice were purchased from the National Cancer
Institute, National Institutes of Health (Bethesda, MD). CXCR3-deficient
mice (CXCR32/2) in the C57BL/6 background (32) were provided by
C. Gerard (Children’s Hospital, Boston, MA) and bred in our facility. OT-I
TCR mice in the C57BL/6 background were obtained from The Jackson
Laboratory (Bar Harbor, ME) and crossed with CXCR32/2mice to gen-
erate CXCR32/2/OT-I mice. CC10-OVA mice in the C57BL/6 background
were generated and maintained in our laboratory (21). Thy1.1+Thy1.2+
double-positive CC10-OVA mice were generated by mating CC10-OVA
with B6.PL-Thy1a/CyJ (The Jackson Laboratory). CD802/2/CD862/2/
CC10-OVA mice were generated by mating CC10-OVA with B6.129S4-
Cd80tm1ShrCd86tm2Shr/J (The Jackson Laboratory). DEREG (Depletion of
Regulatory T cell) mice in C57BL/6 background (40) were crossed with
CC10-OVA mice to generate DEREG/CC10-OVA in our facility. All
protocols were approved by the Massachusetts General Hospital Sub-
committee on Research and Animal Care.
OT-I cell preparation and adoptive transfer
Isolation and preparation of OT-I and CXCR32/2/OT-I CD8+Teff was
performed as described previously (41). Briefly, spleens were harvested
from OT-I TCR transgenic mice, single-cell suspensions were prepared,
and CD8+cells were purified using MACS CD8a MicroBeads kit (Miltenyi
Biotech). Effector OT-I cells were prepared by placing the purified CD8+
OT-I cells in culture for 5 d with irradiated APCs prepared from spleens of
C57BL/6 mice with 700 ng/ml SIINFEKL peptide, 2 mg/ml anti-CD28, 10
ng/ml recombinant IL-2, and 10 ng/ml recombinant IL-12. Effector CD8+
OT-I cells were then resuspended in PBS and injected i.p.
OT-I cell cytotoxicity
Cytotoxicity was measured using a commercially available kit according
to the manufacturer’s protocol (CyToxiLux Plus, OncoImmunin, Gaithers-
CC10-OVA mouse tissue sampling and processing
Animals were sacrificed with a lethal injection of ketamine (100 mg/kg).
The lungs were lavaged with six 0.5-ml aliquots of PBS containing 0.6
mM EDTA. The spleen, thoracic lymph nodes, and inguinal lymph nodes
were removed. The lungs were flushed free of blood by slowly injecting 10
ml PBS into the right ventricle prior to excision and digested for 45 min in
RPMI 1640 with 0.28 Wunsch U/ml Liberase Blendzyme (Roche, Indi-
anapolis, IN) and DNase 30 U/ml (Sigma-Aldrich, St. Louis, MO) at 37˚C.
The digested lungs were then extruded through a mesh strainer.
Tissue was placed into 10% buffered formalin. Multiple paraffin-embedded
5-mm sections were prepared and stained with H&E. The slides were
evaluated by light microscopy.
Flow cytometry and cell sorting
Cells recovered from cell culture, the bronchoalveolar lavage (BAL) fluid,
or single-cell suspensions of lung, lymph node, or spleen were blocked,
stained, and analyzed as previously described (42). The class I tetramer
specific for OT-I cells was obtained from Beckman Coulter (Fullerton,
CA). The staining kit for Foxp3+Treg was obtained from eBioscience (San
Diego, CA). Fluorescently labeled anti-CD3, anti-CD8, anti-Thy1.1, and
anti-Thy1.2 Abs were obtained from BD Pharmingen. Fluorescently la-
beled anti-CXCR3, anti-CCR4, anti-CCR6, and anti-CCR7 were obtained
from BioLegend (San Diego, CA).
Quantitative real-time PCR
RNAwas purified using a purification column (RNeasy; Qiagen, Valencia,
CA). After a DNase step, 1 mg of RNA was converted to cDNA (Applied
Biosystems, Warrington, UK). Specific primers used for sequence de-
tection of message for the CXCL10 (IP-10) gene were 59-GCCGT-
CATTTTCTGCCTCA-39 and 59-CGTCCTTGCGAGAGGGATC-39, for
CXCL9 (MIG) gene were 59-AATGCACGATGCTCCTGCA-39 and 59-
AGGTCTTTGAGGGATTTGTAGTGG-39, for CXCL11 (ITAC) gene
were 59-AATTTACCCGAGTAACGGCTG-39 and 59-ATTATGAGGCGA-
GCTTGCTTG-39, and for the GAPDH gene were 59-GGCAAATTCAA-
underwent amplification in the presence of SYBR Green (Applied Bio-
systems). The reaction was analyzed in real-time during amplification by
the PCR machine (MX-4000; Stratagene, La Jolla, CA).
Lung and spleen from surviving CC10-OVA mice adoptively transferred
with CXCR32/2/OT-I cells for 2 wk were made into single-cell suspen-
sions, and the CXCR32/2/OT-I cells were isolated by cell sorting as re-
sponder cells in two separate experiments. We used class I tetramer to sort
for CXCR32/2/OT-I cells from CC10-OVA mice in the first experiment
and anti-Thy1.2 and anti-Thy1.1 Abs in the second experiment to sort for
Thy1.2+CXCR32/2/OT-I cells from Thy1.1+Thy1.2+double-positive
CC10-OVA mice. In vitro OT-I cells were taken from freshly prepared
effector OT-I cells as described above. Spleen cells from C57BL/6 mice
were used as stimulator cells. Stimulator cells were incubated with or
without SIINFEKL peptide at 700 ng/ml. Responder and stimulator cells
were incubated together in a 96-well plate in triplicate for each group for
2 d at 37˚C. One group also received recombinant IL-2 at 10 ng/ml. [3H]
(0.5 ml) was added to each well and then incubated overnight before the
plate was harvested and read in a scintillation plate counter machine
(TopCount NXT; Packard Bioscience).
Selective Treg depletion
The DEREG mouse is a transgenic mouse carrying a DTR-eGFP transgene
under the control of an additional Foxp3 promoter (40). DEREG/CC10-
OVA mice were i.p. injected with 1 mg diphtheria toxin (EMD Bio-
sciences, San Diego, CA) for 2 consecutive days starting 2 d before Teff
adoptive transfer and a third diphtheria toxin injection 3 d later.
Deficiency of CXCR3 on CD8+Teff diminish mortality and
We previously developed a novel transgenic model of acute lung
rejection where C57BL/6 mice express a membrane-bound form of
chicken egg albumin (OVA) in the airway lining cells of the lung
(CC10-OVA mice) (21). Transfer of 5 3 105in vitro-activated
CD8+Teff with a TCR specific for OVA (isolated from the OT-I
C57BL/6 TCR-transgenic mouse) into CC10-OVA mice induced
The Journal of Immunology6831
airway injury, inflammation, and death within 5–9 d of transfer. In
this study, we evaluated the ability of CXCR3 to mediate Teff
recruitment in the CC10-OVA lung rejection model. CD8+T cells
from the spleens of OT-I and CXCR3-deficient OT-I mice were
isolated and activated in vitro with IL-2 and IL-12 to generate
Teff, as previously described (21, 41). Flow cytometry demon-
strated an equivalent effector phenotype for WT and CXCR3-
deficient OT-I Teff: low CD62L, high CD25, high IFN-g, and
positive perforin expression (Fig. 1A), along with high cytotoxic
ability (Fig. 1B). CC10-OVA mice that received in vitro-activated
CXCR32/2OT-I Teff had a dramatic reduction in mortality
compared with CC10-OVA mice that received WT OT-I Teff (Fig.
1C). Two weeks after Teff adoptive transfer, 91% of CC10-OVA
mice that received CXCR32/2Teff were alive, whereas only 9%
of CC10-OVA mice that received WT Teff survived. Histological
analysis of the lungs 3 d after adoptive transfer of WT OT-I Teff
demonstrated perivascular and peribronchial inflammation (Fig.
1Di, 1Dii). In contrast, mice that received CXCR32/2OT-I Teff
showed minimal inflammation around the lung vasculature and
almost no involvement of the airways (Fig. 1Diii, 1Div). To de-
termine the ligands that mediate the recruitment of Teff through
CXCR3 signaling in our model, we isolated the lungs of CC10-
OVA mice and C57BL/6 controls that received adoptively trans-
ferred OT-I cells 3 d prior and performed quantitative real-time
PCR for CXCR3 ligand expression. As can be seen in Fig. 1E,
CXCL10 and CXCL9 were highly induced (12- and 8-fold, re-
spectively) in the CC10-OVA lungs compared with C57BL/6
control lungs. The expression of these chemokines has been
shown to correlate with T cell recruitment into transplanted organs
during AR (28, 30, 33, 43). CXCL11 RNA expression was absent
in both strains.
CXCR3-deficent Teff have impaired homing into the lung
To compare the trafficking of CXCR32/2Teff to WT Teff in our
mouse model, we performed competitive homing assays (21).
Briefly, CXCR32/2OT-I cells expressing Thy1.2 and WT OT-I
cells expressing Thy1.1 were both adoptively transferred into the
same CC10-OVA mouse congenic for both Thy1.1 and Thy1.2.
This allowed us to track the individual populations of transferred
WT OT-I (Thy1.1+) and CXCR32/2OT-1 (Thy1.2+) cells in the
same recipient mouse as well as distinguish endogenous T cells
(Thy1.1+/Thy1.2+). Analysis of BAL fluid and lung tissue 4 d
after cotransfer revealed a 40% decrease in the accumulation of
CXCR32/2OT-I cells in the lung and BAL compared with WT
OT-I cells (Fig. 2A, 2B). In contrast, the peripheral organs, such as
the spleen and inguinal lymph nodes, contained 2-fold and 1.7-
fold more CXCR32/2OT-I cells than WT OT-I, respectively.
These data indicate that CXCR3 plays a role in Teff trafficking
into the lung and that inhibition of CXCR3-mediated Teff re-
cruitment in this model can reduce mortality.
Teff are anergic in the lungs of survivors
The competitivehoming assay revealedthat some CXCR32/2OT-I
cells still reached the airways and accumulated in surviving mice
(Fig. 2B). We therefore transferred only CXCR32/2OT-I Teff into
CC10-OVA mice and analyzed the BAL of surviving mice 2 wk
after adoptive transfer for the presence of these transferred Teff.
As a secondary method of identifying these adoptively transferred
CXCR32/2OT-I Teff, we used fluorochrome-conjugated class I
tetramers specific for the OT-I TCR and anti-CD3. This analysis
revealed that the Teff were still present in the airways (Fig. 2C)
but were apparently not causing overt signs of pulmonary damage.
OT-I into CC10-OVA mice. A, In vitro-activated
CD8+cells from CXCR32/2or WT OT-I mice
stained for activation markers (CD62L, CD25,
IFN-g, and perforin). B, Cytotoxicity assay of
in vitro-activated CXCR32/2or WT OT-I cells
using the CyToxiLux kit with EL-4 cells as tar-
gets. C, Mortality of CC10-OVA transgenic mice
injected with WT OT-I Teff or CXCR32/2OT-I
Teff (n = 11 per group). Curves were significantly
different by log-rank test. D, Representative his-
tologies of lungs from CC10-OVA transgenic mice
3 d after adoptive transfer of WT OT-I Teff stained
with H&E (i, ii) or CXCR32/2OT-I Teff (iii, iv).
Original magnification: low power at 34 (i, iii)
and high power at 340 (ii, iv). Black arrows in-
dicate apparent perivascular and peribronchial in-
flammation. E, CXCR3 ligand expression in the
lungs of C57BL/6 and CC10-OVA mice 3 d after
determined by quantitative real-time PCR.
Reduced mortality and pulmonary
6832REDUCING Teff TRAFFICKING INDUCES Treg AND LUNG TOLERANCE
We then isolated the CXCR32/2OT-I cells from the lungs and
spleens of surviving CC10-OVA mice 2 wk after adoptive transfer
using FACS and determined their ability to proliferate to OVA
restimulation in vitro. CXCR32/2OT-I cells isolated from the
lung showed a 3.3-fold decrease in OVA-induced proliferation
compared with cells isolated from the spleen of the same recipient
mouse, as well as from in vitro-activated WT OT-I cells (Fig. 2D).
However, the addition of exogenous IL-2 to CXCR32/2Teff
isolated from the lung dramatically overcame the proliferative
defect (Fig. 2D). These data demonstrate that CXCR32/2OT-I
cells in the lungs of surviving mice were unresponsive to their
target Ag and that the effect was specific to the lungs.
Teff induce Treg in the lungs
Analysis of surviving CC10-OVA mice 2 wk after adoptive transfer
of CXCR32/2OT-I cells revealed a 2-fold increase in FOXP3+
Treg in the lungs compared with WT C57BL/6 mice that also
received the CXCR32/2OT-I cells or compared with untreated
CC10-OVA mice (Fig. 3A). This increase in Treg was specific to
the lung, as there was no difference in the number of FOXP3+
Treg in the spleen among the different groups (Fig. 3A). We also
examined an earlier time point after the adoptive transfer of
CXCR32/2and WT OT-I Teff. We reasoned that if CXCR32/2
Teff that entered the lung induced Treg accumulation in the lung,
then CXCR3+/+Teff that entered the lung might also induce Treg
accumulation. We found a 2-fold increase in CD4+FOXP3+cells
in the lung 3–4 d after WT OT-I Teff transfer compared with
untreated mice (Fig. 3B). In contrast, the lungs of CC10-OVA
mice that received CXCR32/2OT-I Teff showed no increase in
Treg at this early period. Consistent with the specificity for the
lung, the spleen showed no early increase in Treg after either WT
or CXCR32/2OT-I transfer compared with the control (Fig. 3B).
To determine if there is similar chemokine signaling for the
recruitment of Teff and Treg in the lung, we ascertained the che-
lung and spleen of CC10-OVA or C57BL/6 mice 3 d after OT-I
adoptive transfer by flow cytometry (Fig. 3C). OT-I Teff re-
covered from the spleen expressed high levels of CXCR3 and low
levels of CCR7, similar to in vitro-generated Teff prior to in-
jection, as we have previously reported (44). In contrast, Treg
recovered from the spleen expressed low levels of CXCR3 and
high levels of CCR7. CCR4 and CCR6 were expressed to similar
levels on Teff and Treg recovered from the spleen. In the lung,
Teff recovered from C57BL/6 mice also expressed high levels of
CXCR3. However, CXCR3 was downregulated (4.5-fold) on Teff
recovered from the lungs of CC10-OVA mice compared with
C57BL/6 mice. Treg recovered from the lungs of C57BL/6 and
CC10-OVA mice showed low expression of CXCR3 with levels in
the CC10-OVA mice 1.9-fold less than in C57BL/6 mice. Both
Teff and Treg in the lung of CC10-OVA mice showed a slight
increase in CCR6 expression (1.9- and 1.7-fold, respectively)
compared with those from C57BL/6 mice. These data suggest that
the recruitment of Teff and Treg are likely controlled by different
chemokine pathways in this model.
Lung rejections depend on the number of Teff recruited to the
We next determined if more CXCR32/2Teff adoptively trans-
ferred into CC10-OVA mice would result in an increase in their
accumulation in the lung and overcome regulatory mechanisms
and induce rejection. We found that a 3-fold increase in the
number of CXCR32/2Teff did indeed induce 100% mortality
compared with 30% mortality observed with the standard dose of
5 3 105cells (Fig. 4A). As a further means to determine if the
sponsiveness of CXCR32/2OT-I cells in the
airways. A, Representative flow cytometry
of CD3+lymphocytes revealing WT OT-I
(Thy1.1+) and CXCR32/2OT-I (Thy1.2+)
Teff recruitment into the lung and BAL 4 d
cytometry data of Thy1.1+WT OT-I and
Thy1.2+CXCR32/2OT-I recruited into the
lung, BAL, spleen, inguinal lymph node
(iLN), and thoracic lymph node (tLN) 4 d
after cotransfer into CC10-OVA mice (n = 4
mice). *p , 0.04. C, Representative flow
cytometry of BAL for OT-I cells 2 wk after
adoptive transfer into CC10-OVA mice. The
transferred cells are identified by tetramer
staining specific for the OT-I TCR and anti-
CD3 staining. D, [3H] proliferation assay of
recovered CXCR32/2OT-I Teff isolated from
the lung and spleen of CC10-OVA survivors 2
wk after adoptive transfer by cell flow sorter
and activated WT OT-I cells from 5-d culture.
The cells were, or were not, restimulated with
OVA peptide and exogenous rIL-2.
Decreased recruitment and re-
Summary of flow
The Journal of Immunology 6833
number of Teff reaching the lung was a crucial determinant for
organ rejection, we reversed our approach and asked if reducing
the number of WT OT-I Teff adoptively transferred into the CC10-
OVA mice would prevent death. As seen in Fig. 4B, the standard
number of 5 3 105cells predictively resulted in 100% mortality
6 d after transfer. Lowering the number of transferred Teff by
more than half to 2 3 105cells delayed death to 9 d after transfer.
Further reduction to 1 3 105cells, one-fifth the standard number
of transferred cells, resulted in only 14% mortality. We next de-
termined if the transfer of lower numbers of WT Teff also induced
Treg in surviving mice. The lungs from surviving CC10-OVA
mice showed nearly a 3-fold increase in FOXP3+Treg com-
pared with their WT C57BL/6 controls (Fig. 4C), indicating that at
one-fifth the standard dose, WT OT-I cells reached the lungs and
induced the accumulation of Treg. The draining thoracic lymph
nodes from surviving CC10-OVA mice also showed an increase in
the FOXP3+cells by 1.5-fold. In contrast, as was seen with the
transfer of CXCR32/2Teff, the spleen and inguinal lymph nodes
of CC10-OVA mice that received 1 3 105WT OT-I cells did not
show any difference in FOXP3+cells compared with their C57BL/
6 controls (Fig. 4C).
Treg are essential to prevent acute lung rejection and induce
We demonstrated that a low dose of effector CD8+OT-I cells in the
CC10-OVA mice induced the accumulation of Treg specifically
in the lung. To demonstrate that these regulatory cells were truly
protective against Teff-induced rejection, we took three comple-
mentary approaches: 1) determined if the increase in Treg in
CC10-OVA mice after low-dose Teff transfer, as shown in Fig. 4B
of CC10-OVA survivors. A, Summary graphs of
the percentage of Foxp3+cells within gated
CD4+lymphocytes in the lungs and spleens of
untreated CC10-OVA mice and WT C57BL/6
mice and CC10-OVA survivors 2 wk after in-
jection with CXCR32/2OT-I Teff. B, Summary
graphs of the percentage of Foxp3+cells in the
lungs and spleens of CC10-OVA mice 3–4 d after
adoptive transfer of WT or CXCR32/2OT-I Teff
and untreated CC10-OVA mice. C, Representa-
tive flow cytometry histograms of chemokine
receptor cell surface expression on CD8+OT-I
Teff and CD4+Foxp3+Treg recovered from the
lungs and spleens of CC10-OVA mice 3 d after
adoptive transfer of WT OT-I (Thy1.1+) Teff.
Summary graphs presented below histograms ex-
hibit the mean percentage and SD of CD8+
Thy1.1+OT-I cells and CD4+Foxp3+Treg from
CC10-OVA or C57BL/6 mice expressing CXCR3,
CCR4, CCR6, or CCR7 chemokine receptors
(n = 6 CC10-OVA, 3 C57BL/6). *p , 0.05.
Foxp3+Treg increased in the lungs
6834 REDUCING Teff TRAFFICKING INDUCES Treg AND LUNG TOLERANCE
and 4C, would prevent rejection from a subsequent normally le-
thal Teff dose; 2) used Treg-deficient CD802/2/CD862/2/CC10-
OVA mice; and 3) used selective Treg-depletable DEREG/CC10-
OVA mice to specifically deplete Treg.
In our first approach, CC10-OVA mice were adoptively trans-
ferred with 1 3 105WT OT-I cells, as described above, but after 2
wk a second dose of WT OT-I cells that would normally be fatal
was given to the same recipient CC10-OVA mice (Fig. 5A). We
hypothesized that the large increase in FOXP3+Treg that accu-
mulated in the lung after the low-dose OT-I transfer would mod-
ulate the effector functions of subsequent OT-I cells given to the
CC10-OVA mice and prevent fatal pulmonary inflammation.
Whereas only 20% of the control CC10-OVA mice given 2.5 3
105OT-I cells survived after 7 d, 100% of the CC10-OVA mice
first given 1 3 105OT-I cells survived for more than 30 d after
receiving a second dose of 2.5 3 105OT-I cells (Fig. 5A). We next
determined if a higher number of OT-I cells in the second dose
could also be prevented from inducing mortality. As can be seen in
Fig. 5A, 100% of the control mice given 5 3 105OT-I cells died,
as previously shown (Figs. 1C, 4B), but the number of deaths was
dramatically reduced to 14% when the mice were first given a low
dose of 1 3 105OT-I cells before the standard dose of 5 3 105
OT-I cells. Greater than 25% of the CD8+cells in the lungs of the
survivors were found to be the adoptively transferred OT-I Teff
(Supplemental Fig. 1). These results demonstrate that a nonlethal
dose of WT Teff was protective against a subsequently higher,
normally lethal dose of WT Teff.
In our second approach, we used CC10-OVA mice lacking the
costimulatory molecules CD80 and CD86 as recipient mice.
CD802/2/CD862/2mice have a profound deficiency in FOXP3+
Treg (45) and they were mated with our CC10-OVA mice to
generate CD802/2/CD862/2/CC10-OVA mice. In the spleens of
these mice, ,1% of CD4+T cells are FOXP3+Treg, compared
with 5–8% of CD4+T cells in CC10-OVA mice (Supplemental
Fig. 2). Adoptive transfer of 1 3 105OT-I Teff into Treg-deficient
CD802/2/CD862/2/CC10-OVA mice resulted in 80% mortality
within 7 d, whereas transfer of the same number of Teff into
CC10-OVA mice resulted in 20% mortality (Fig. 5B).
We hypothesized that it was the lack of Treg in CD802/2/
CD862/2/CC10-OVA mice that prevented the suppression of OT-I
Teff pathogenicity in the lungs. To confirm this hypothesis, our
third approach was to breed the selective Treg-depletable DEREG
mouse (40) to our CC10-OVA mouse. Treatment of DEREG mice
with two consecutive doses of diphtheria toxin (DT) selectively
depleted up to 93% of the FOXP3+Treg in the spleen, lymph
nodes, and lung 1 d after the last treatment (Supplemental Fig. 3).
DEREG/CC10-OVA mice were treated with the same two con-
secutive doses of DT, along with one more dose 3 d after the
adoptive transfer of 1 3 105OT-I Teff. As hypothesized, all of the
DT-treated DEREG/CC10-OVA mice died within 7 d (Fig. 5C),
whereas, the Treg-containing CC10-OVA mice showed only 37%
mortality after receiving 1 3 105OT-I Teff. Treatment of DEREG/
CC10-OVA mice with DT alone showed no mortality (data not
shown). In other experiments, DEREG/CC10-OVA and CC10-
OVA mice were injected with low-dose OT-I cells, and survivors
were then treated with DT for 2 consecutive days. Two days later,
lungs and spleens were harvested and analyzed for Treg and Teff
numbers (Fig. 5D, 5E). As expected, DT treatment eliminated
Treg from the lungs and spleens of DEREG/CC10-OVA mice but
not from CC10-OVA mice (Fig. 5D). Of note, along with this Treg
number of Teff recruited to airways. A, Mortality of
CC10-OVA mice injected with CXCR32/2OT-I
Teff at doses of 0.5 3 106or 1.5 3 106cells.
Curves were significantly different by log-rank test
(p = 0.0002). Mortality curve of WT C57BL/6
mice injected with 1.5 3 106CXCR32/2OT-I Teff
was not significantly different from that of CC10-
OVA mice injected with 0.5 3 106CXCR32/2OT-
I cells by log-rank test. B, Mortality of CC10-OVA
mice adoptively transferred with WT OT-I Teff
at three different doses: 0.5 3 106, 0.2 3 106, and
0.1 3 106. The lowest-dose curve was significantly
different from that of the other two curves by log-
rank test (p , 0.0001). C, Summary graphs of the
percentage of Foxp3+cells within gated CD4+
lymphocytes from the lung, spleen, thoracic and
inguinal lymph nodes of C57BL/6 and CC10-OVA
mice 2 wk after injection with 0.1 3 106WT OT-I
CC10-OVA survival dependent on
The Journal of Immunology 6835
depletion, DT treatment increased the number of OT-I cells found
in the lungs of DEREG/CC10-OVA mice by 7-fold compared with
those found in CC10-OVA mice (Fig. 5E). These data are con-
sistent with our hypothesis that Treg can actively suppress OT-I
Teff recruited into the lung to achieve tolerance. Together, these
results demonstrate that FOXP3+Treg in CC10-OVA mice are
able to suppress the cytopathic activity of Teff that reach the lung
up to a certain threshold.
Using a transgenic mouse model of lung rejection, we have de-
lineated a role for CXCR3 specifically on CD8+Teff in the re-
jection process. In so doing, we have found that decreasing the
number of Teff recruited into the lung allowed endogenous reg-
ulatory mechanisms to be activated by these Teff and induce
organ-specific tolerance. Deletion of the CXCR3 chemokine re-
ceptor pathway on Teff did not completely eliminate their traf-
ficking into the lungs; however, this partial inhibition was
sufficient to allow for the effective generation of active tolerance.
Thus, even partial interruption of Teff recruitment into the target
tissue can generate a graft-specific microenvironment conducive
to Treg generation and function.
Animal models of transplantation have increased our under-
standing of the mechanisms underlying rejection and chronic al-
lograft dysfunction. Well-established murine models of cardiac,
renal, and skin transplantation have been used to determine the role
of several chemokines in transplantation (46). However, because
lung transplantation in small animals is technically difficult, an
ideal murine model of lung transplantation does not exist. We
developed the CC10-OVA transgenic mouse (21) to use as a model
for acute lung transplant rejection. Adoptive transfer of activated
CD8+OT-I cells specific for the OVA peptide leads to respiratory
distress and death within 7 d. In this transgenic mouse model,
OVA is membrane bound on the airway epithelium, resulting in
CD8+T cell-mediated injury to the airway lining, which closely
mimics the pathophysiology of AR. The model exhibits significant
perivascular and peribronchial inflammation typically seen in AR
and serves as a proof-of-concept for specifically examining Ag-
specific effector CD8+T cells targeted against a specific organ.
Similar transgenic mouse models, such as the RIP-OVA mouse,
have been successfully used to study T cell tolerance, tissue
damage, and T cell trafficking (47, 48).
Previous studies in humans and animal models of transplan-
tation have identified CXCR3 and two of its ligands, CXCL9 and
plantation (28, 30, 32, 34, 43, 49). Recently, the role of CXCR3 in
organ rejection has become less clear with reports indicating that
this chemokine receptor pathway is not essential for the rejection
process (35, 36). These prior studies used either CXCR3-deficient
recipients or CXCR3 antagonists to interrogate CXCR3 function.
This type of experimental design would render CXCR3 non-
functional on all cell types, including FOXP3+Treg, which have
recently been shown capable of expressing CXCR3 (38, 50, 51).
However, the design of our study enabled us to isolate specifically
an important role for CXCR3 on Ag-specific CD8+Teff in induc-
ing pulmonary AR. We demonstrate that CXCR3 contributes
importantly to the ability of Teff to home to the lung and that
this CXCR3-dependent Teff trafficking also contributes to pul-
monary inflammation and injury and resultant mortality in a
model of AR.
lung rejection. A, Mortality of CC10-OVA mice
injected i.p. with serial doses of WT OT-I Teff;
first low dose of 1 3 105cells was given 2 wk
before the second dose of 2.5 3 105or 5.0 3
105cells on day 0. Control CC10-OVA mice
were given a single dose of 2.5 3 105or 5.0 3
105OT-I Teff on day 0. B, Mortality of CC10-
OVA and CD802/2CD862/2/CC10-OVA litter-
mates injected with 1 3 105OT-I Teff. C,
Mortality of DEREG/CC10-OVA mice treated
with DT to deplete Treg and injected with 1 3
105OT-I Teff on day 0. Littermate CC10-OVA
control mice were injected with either 1 3 105
or 5 3 105OT-I Teff on day 0. D and E, CC10-
OVA and DEREG/CC10-OVA littermates were
injected i.p. with low-dose WT in vitro-activated
OT-I (Thy1.1+) cells on day 0. On day 10, sur-
vivors of both strains were treated with DT for 2
consecutive days. On day 13, spleens and lungs
were harvested and pooled from two CC10-OVA
and two DEREG/CC10-OVA mice. D, Flow
cytometry for CD4+FOXP3+cells gated from
lymphocytes after DT treatment. E, Flow cy-
tometry for Thy1.1+OT-I cells gated from CD8+
Resident Treg required to prevent
6836REDUCING Teff TRAFFICKING INDUCES Treg AND LUNG TOLERANCE
We found that Teff recovered from the lungs of CC10-OVA mice
have markedly decreased surface expression of CXCR3 compared
with Teff recovered from the spleens of CC10-OVA mice or from
the lungs of C57BL/6 mice. We believe that these data indicate that
CXCR3 is specifically downregulated on Teff after encounter with
its ligands in the lung and support a functional role for CXCR3 in
the model. Previous studies have found that CXCR3 ligands induce
CXCR3 internalization in vitro as well as in vivo upon entering the
lung where high ligand levels were found (52, 53). In contrast to
Teff, Treg recovered from lungs and spleens of CC10-OVA and
C57BL/6 mice did not express high levels of CXCR3 in any tissue
compartment. Instead, Treg expressed high levels of the lymph
node-homing chemokine receptor CCR7 as well as moderate
levels of CCR6. The large decrease in CCR7 expression on Treg
isolated from the lungs of CC10-OVA mice compared with that of
those from C57BL/6 mice may indicate the activation of endog-
enous naive Treg into effector Treg in the inflamed lung (54), as
such decrease was not seen in the spleen.
Teff and FOXP3+Treg. The net outcome of the pro- and anti-
inflammatory activities mediated by these cells appears to be an
important determinant of allograft tolerance versus allograft re-
jection. We found that early in the rejection process, Teff re-
cruitment into the lung induced an increase in the number of
FOXP3+Treg specifically in the lung (Fig. 3B). However, the
suppressive activity of these regulatory cells was not sufficient to
inhibit the inflammatory activities of Teff recruited into the lung,
and mice ultimately died of acute lung rejection 3–4 d later (Fig.
1C). However, when the number of Teff reaching the lung was
decreased by either CXCR3 deficiency or the transfer of fewer WT
Teff (Fig. 4), mice were able to survive even with residual Teff
present in the lungs as these Teff were now rendered tolerant (Fig.
2D). These data are consistent with a study that found that cardiac
allograft survival was prolonged by blocking CXCR3 and CCR5,
which wasassociated withanincrease inFOXP3+Treginthegrafts
(55). It is becoming apparent that Treg are like other T cell subsets
in that they develop during an immune response (56). Recent
studies have shown that Treg can be present simultaneously with
Teff in inflamed tissues and serve to balance the toxic effects of
microbicidal cytokines (57, 58). The results from our study show
that the same mechanism can also be applied to suppress the organ-
rejecting activity of CD8+Teff.
Treg are now recognized as a fundamental component in the
development and maintenance of transplantation tolerance as they
protect against pathogenic Teff (56, 59). Consistent with this
concept, the data from our model of CD8-mediated rejection
suggest that an increase in lung Treg or a reduction in Teff can
limit lung injury and enhance survival. Notably, the introduction
of Teff into the lung was associated with enhanced accumulation
of Treg specifically in the lung. This increase in Treg may be
secondary to the burst of IL-2 produced by activated Teff in the
inflamed tissue (60), as IL-2 has been found to be crucial in the
proliferation and maintenance of Treg (61, 62). Treg have also
been shown to expand quickly after immune priming before their
suppressive properties become apparent (63), and Teff have been
shown to alter gene transcriptions in Treg (64, 65). Our study adds
to these findings by demonstrating that Teff can boost the function
of Treg in vivo.
Rejection and death occur in our model when a certain threshold
of Teff reaching the lung is reached and the tolerogenic Treg/Teff
ratio is no longer maintained. IL-2 is not only crucial for Tregbut is
also an important growth factor for the Teff population. As more
Teff accumulate in the lung, they may out-compete Treg for
a limited pool of IL-2 to a point where Treg can no longer be
sustained, and rejection is inevitable. In support of this model,
a recent study of mice infected with Toxoplasma gondii demon-
strated that a hyperimmune Th1 mucosal immune response could
lead to collapse of mucosal Treg, resulting in markedly increased
mucosal immunopathology (66). Thus, an overexuberant organ-
specific Teff response appears to be detrimental to the survival and
function of Treg in that organ.
In conclusion, our data demonstrate that inhibition of CXCR3
on Teff may be therapeutically beneficial by inhibiting enough
pathogenic Teff recruitment into the allograft to allow for the
inflammation-induced recruitment and expansion of FOXP3+Treg
selectively in the graft to be effective at suppressing the rejection
process. Our data also suggest that the complete inhibition of
allospecific T cell responses may not be required to induce
transplantation tolerance and that allospecific Teff may in fact
induce Treg expansion in the graft. Thus, modulating the che-
moattractant pathways that participate in the homing of Teff
without also inhibiting the resultant accumulation of Treg may
be a novel approach to prevent graft rejection and induce trans-
The authors have no financial conflicts of interest.
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6838REDUCING Teff TRAFFICKING INDUCES Treg AND LUNG TOLERANCE