The Journal of Clinical Investigation http://www.jci.org
Dendritic cells tolerized with adenosine A2AR
agonist attenuate acute kidney injury
Li Li,1,2 Liping Huang,1,2 Hong Ye,1,2 Steven P. Song,1,2 Amandeep Bajwa,1,2 Sang Ju Lee,1,2
Emily K. Moser,1,2 Katarzyna Jaworska,1,2 Gilbert R. Kinsey,1,2 Yuan J. Day,3 Joel Linden,4
Peter I. Lobo,1,2 Diane L. Rosin,2,5 and Mark D. Okusa1,2
1Department of Medicine and 2Center for Immunity, Inflammation and Regenerative Medicine, University of Virginia, Charlottesville, Virginia, USA.
3Department of Anesthesiology, Chang Gung Memorial Hospital, Taipei, Taiwan. 4Division of Inflammation Biology,
La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. 5Department of Pharmacology, University of Virginia, Charlottesville, Virginia, USA.
DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/
reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important anti-
inflammatory molecule in tissue inflammation, and adenosine 2A receptor (A2AR) agonists protect kidneys
from IRI through their actions on leukocytes. In this study, we showed that mice with A2AR-deficient DCs are
more susceptible to kidney IRI and are not protected from injury by A2AR agonists. In addition, administration
of DCs treated ex vivo with an A2AR agonist protected the kidneys of WT mice from IRI by suppressing NKT
production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation.
A2AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A2AR–induced
tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to
attenuate organ IRI.
Kidney DCs, residing in the interstitial extracellular compart-
ment, are professional APCs and play a critical role in initiating
an early immune response against pathogens as well as maintain-
ing immunological tolerance to self antigens. DCs are activated
by danger-associated molecular patterns (DAMPS) or pathogen-
associated molecular patterns (PAMPS) (1). In the presence of
PAMPS or DAMPS, DCs are key initiators, potentiators, and effec-
tors of the innate immune system, which comprises neutrophils,
monocytes/macrophages, DCs, NK cells, and NKT cells, in kidney
ischemia/reperfusion injury (IRI) and induce injury either directly
or through inflammatory signals (2).
NKT cells represent a subset of innate-like lymphocytes that
share receptor structures and functions with both conventional
T cells and NK cells. NKT cells typically recognize endogenous or
exogenous glycolipid antigens bound by the MHC class I–like pro-
tein CD1d on APCs. Type I NKT cells express an invariant TCR-α
chain encoded by a Vα14-Jα18 rearrangement in mice, whereas the
type II NKT cells possess a diverse TCR. CD1d-restricted type I
NKT cells, but not type II NKT cells, recognize the strong agonist/
glycolipid α-galactosylceramide (αGC), originally isolated from a
marine sponge (3). A unique property of NKT cells is their capacity
to rapidly produce both Th1-type (IL-2 and IFN-γ) and Th2-type
(IL-4 and IL-10) cytokines upon TCR engagement.
NKT cells initiate inflammation following IRI in liver, lung, and/or
kidney (4, 5). By studying NKT cell–deficient mice and by blocking
DC-NKT cell interaction, we demonstrated that CD1d-restricted
NKT cells are necessary for kidney IRI. NKT cells are activated
rapidly after IRI, and the initial NKT cell response is followed by
a secondary activation of other immune cells and by cytokine pro-
duction, which crucially influences the subsequent inflammatory
cascade in kidney IRI (6). Modulating NKT cell function is a strate-
gy used in the treatment of autoimmune diseases and cancer (7, 8)
and in the generation of various vaccines (9). Therefore, specifical-
ly modulating NKT cell function has broad clinical significance.
Although DCs contribute to immune activation, they also induce
tolerance. Tolerogenic DCs have been used as potential therapeu-
tic tools and represent a new and promising immunotherapeutic
approach for ameliorating or preventing graft rejection or treating
autoimmune disorders, cancers, and other serious conditions (10).
Immature myeloid DCs that express low surface levels of MHC
class II and costimulatory molecules induce T cell tolerance, where-
as mature myeloid DCs, which express much higher levels of these
molecules, induce T cell immunity. BM-derived DCs (BMDCs)
have been rendered tolerogenic by exposure to cytokines, growth
factors, or pharmacological mediators, or by genetic engineering
(11, 12). Tolerogenic DCs can produce T cell death, T cell anergy,
or Treg expansion and can regulate autoreactive or alloreactive T
cell responses and promote or restore antigen-specific tolerance in
experimental animal models (13). Tolerogenic DCs of either donor
or host origin can promote transplant tolerance induction (14).
Kidney-resident DCs reside in the interstitium throughout
the kidney (15, 16) in close apposition to tubular epithelial cells,
endothelial cells, macrophages, and fibroblasts (17), where they
can respond to changes in the local microenvironment. DC pheno-
type is likely determined by direct cell contact or through soluble
mediators. For example, fibroblasts express an abundance of CD73
(17), the enzyme that catalyzes the final step in the breakdown of
ATP/ADP to adenosine. Adenosine concentrations increase dra-
matically in inflamed and remodeling tissues (18, 19). Extracellular
adenosine binds to adenosine receptors (ARs), the A1AR, A2AR,
A2BR, and A3R (20). Activation of adenosine 2A receptor (A2AR),
a Gs-coupled receptor, increases intracellular cAMP, which is a
potent inhibitor of the NF-κB pathway downstream of immuno-
receptors (21) and therefore may contribute to the antiinflamma-
tory effects of A2AR agonists (4, 22). Ohta et al. showed that A2AR
agonists attenuate tissue-specific and systemic inflammation (23);
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. doi:10.1172/JCI63170.
2 The Journal of Clinical Investigation http://www.jci.org
this antiinflammatory effect is mediated through inhibition of
TLR-induced transcription of proinflammatory cytokines (24).
We also showed that A2AR agonists ameliorate kidney IRI through
effects on BM-derived cells (25, 26) and that NKT cell activation
is necessary for the innate immune response in kidney IRI (4, 6).
Our data showed that activation of A2AR expressed either on DCs
or T cells suppresses the immune response in allograft rejection
(27). In addition, activation of A2ARs mediates inhibition of T cell
proliferation and expansion and NK cell cytotoxicity (28, 29). Even
short-term exposure to adenosine is sufficient to inhibit TCR-
triggered effector CD4+ and CD8+ T cell functions (30), which by
demonstrating a memory effect of activation of A2AR on immune
cells, provides support for the concept of prolonged effect of adop-
tive transfer of A2AR agonist–pretreated DCs in the current study.
In the current study, we hypothesized that A2AR agonists, e.g.,
ATL313, act on DCs loaded with NKT cell antigen αGC (DCs-αGC)
to induce tolerance, and these tolerogenic DCs may be admin-
istered to mice to block NKT cell activation and prevent acute
kidney injury (AKI). Conceptually this cell-based therapeutic
approach used in the treatment of cancer and autoimmune dis-
ease as well as AKI may be useful in minimizing side effects of
systemically administered drugs and in targeting specific immune
cells in kidney IRI. Our data showed that these ATL313-treated
antigen-pulsed DCs (DCs-αGC-ATL313) have regulatory proper-
ties and protected kidney from IRI in vivo by suppressing IFN-γ
production. ATL313 had no effect on DC CD1d/glycolipid com-
plex formation and therefore didn’t interfere with antigen pre-
sentation and NKT cell recognition. However, ATL313 downregu-
lated positive and upregulated negative costimulatory molecule
expression on DCs-αGC. IL-10 produced from systemic sources
contributed to kidney protection. We found that IL-10 can be
produced from spleen B220+ B cells and CD11c+ cells. Our study
indicates that (a) renal interstitial DC phenotype is determined
in part by the expression of A2ARs and activation by selective ago-
nists reduces IRI and (b) ex vivo A2AR agonist–generated tolero-
genic DCs provide what we believe to be a new therapeutic strat-
egy in the prevention of AKI by suppressing NKT cell–mediated
innate immune responses.
Activation of A2AR expressed on CD11c+ DCs is important for kidney IRI.
We have shown that activation of A2AR on BM-derived cells pro-
tects kidneys from IRI (26). Using CD11c-DTR transgenic mice
and administration of diphtheria toxin to selectively deplete DCs,
we have also shown that DCs are necessary for the development of
injury in the kidney following IR (31). We now show that sort-puri-
fied CD11c+ kidney DCs express all 4 subtypes of ARs (A1R, A2AR,
A2BR, and A3R), but mRNA expression of these receptors was not
changed after ischemia and 24 hours of reperfusion (Supplemental
Figure 1; supplemental material available online with this article;
doi:10.1172/JCI63170DS1). To further examine the role of A2AR
on DCs, we generated mice lacking 1 or both A2AR alleles only on
CD11c+ DCs and performed subthreshold kidney IRI; conditional
deletion of A2ARs using the Adora2afl/fl mice has been described (32),
and genotyping of the DC A2AR KO has been confirmed (Y.J. Day
and J. Linden, unpublished results). The small increase in injury
(“mild injury” or subthreshold IRI, produced by a modest period
of ischemia), indicated by elevated plasma creatinine in CD11c-Cre
(WT) mice, was markedly increased in CD11c-CreAdora2afl/WT and
CD11c-CreAdora2afl/fl mice (Figure 1), which is similar to our prior
results in global Adora2a–/– mice, and suggested that absence of the
A2AR-dependent protective effect of endogenous adenosine renders
A2AR-deficient mice more susceptible to injury (26). In addition,
the A2AR agonist ATL313 was not protective in either CD11c-Cre-
Adora2afl/WT or CD11c-CreAdora2afl/fl mice, which is similar to the
ATL313 effect on global Adora2a–/– mice (26). Thus the data indi-
cate that DC A2ARs activated by endogenously released adenosine
or selective A2AR agonists mediate tissue protection.
Activation of A2AR signaling by ATL313 blocks NKT cell activation in kid-
ney IRI. Our previous findings showed the proinflammatory role of
IFN-γ production from NKT cells in IRI (6, 33) and that targeting
NKT cell suppression is an important strategy for inhibiting IRI.
The tissue-protective effects of A2AR activation could involve mod-
ulation of NKT cell activation pathways. ATL146e (10 ng/kg/min,
s.c.), another selective A2AR agonist with properties similar to those
of ATL313, significantly decreased IFN-γ−producing NKT cell
migration to the inflamed kidney following 24-hour reperfusion
compared with vehicle-treated control IRI mice (Figure 2A).
To investigate the effect of A2AR stimulation on DC-NKT cell inter-
action, BMDCs primed with the glycolipid antigen αGC (DCs-αGC)
were administered to mice to activate NKT cells before subthresh-
old kidney IRI, a paradigm known to contribute to kidney IRI (34).
ATL313 (1 ng/kg/min, s.c.) was delivered by osmotic mini-pump
to WT mice at the onset of DC-αGC adoptive transfer. To restrict
the effect of A2AR agonist stimulation to DC-αGC-NKT interaction
prior to surgery, ATL313-loaded mini-pumps were removed after 2
days, and mice were then subjected to subthreshold (mild) injury.
The increase in plasma creatinine induced in the DC-αGC control
group following subthreshold injury was markedly decreased in the
ATL313-treated DC-αGC group (Figure 2B). These results indicate
that activation of the A2AR suppressed DC-αGC–mediated NKT cell
activation in vivo, and blocking this activation protected kidneys
from IRI. However, the contribution of A2AR signaling on other cells
cannot be excluded, and the next set of experiments explored the
direct effect of ATL313 on DC-NKT cell interactions.
Activation of A2AR expressed on CD11c+ DCs is important for kidney
IRI. A2AR agonist ATL313 (10 ng/kg/min, osmotic mini-pump, s.c.) was
administered to mice 24 hours prior to kidney IRI surgery. Following
26 minutes of kidney ischemia and 24 hours of kidney reperfusion,
plasma creatinine levels were measured from vehicle- and ATL313-pre-
treated CD11c-Cre, CD11c-CreAdora2afl/WT, and CD11c-CreAdora2afl/fl
mice. Kidney pedicles of sham-operated animals were exposed but not
clamped. Values are mean ± SEM. n = 3–7. **P < 0.01.
The Journal of Clinical Investigation http://www.jci.org
DCs-αGC-ATL313 protect kidneys from IRI. Based on the above
results, we hypothesized that A2AR agonists may tolerize DCs and
that protection from kidney injury may result from reduced DC-
mediated NKT cell activation in the presence of tolerized DCs
loaded with NKT cell agonist αGC. To isolate the A2AR effect on
DCs, we treated DCs with ATL313 during priming with αGC ex
vivo. WT DCs-αGC were pretreated with ATL313 (1 nM; WT DCs-
αGC-ATL313) and washed extensively (to remove any residual
ATL313); an optimal dose of 0.5 × 106 cells was adoptively trans-
ferred to naive WT mice 2 days prior to kidney surgery to allow
Suppression of DC-NKT interaction in vivo by A2AR activation or by treatment of mice with A2AR-activated αGC-loaded BMDCs protects kidneys
from IRI. (A) WT mice were pretreated with A2AR agonist ATL146e (10 ng/kg/min, osmotic mini-pump, s.c.) 24 hours prior to kidney IRI sur-
gery. IFN-γ–producing live CD45+7-AAD–CD1d-tetramer+TCRβ+ NKT cell number was measured by FACS 24 hours after sham surgery or after
28 minutes of kidney ischemia. Values are mean ± SEM. n = 3–5. (B) Pretreatment of WT mice with vehicle (saline) or the A2AR agonist ATL313
(1 ng/kg/min, osmotic mini-pump, s.c.) was initiated at the onset of adoptive transfer of BMDCs loaded with αGC (DC-αGC) or vehicle (DC);
mini-pumps were removed 2 days later, and then mice were subjected to sham surgery or subthreshold (26 minutes ischemia) mild IRI. Plasma
creatinine was measured 24 hours after sham or IRI surgery. Values are mean ± SEM. n = 2–4. (C and D) WT or Adora2a–/– DCs were primed ex
vivo with vehicle (DC) or αGC in the presence (DC-αGC-ATL313) or absence (DC-αGC) of ATL313 (1 nM), incubated for 2.5 days, washed, and
adoptively transferred to WT mice 2 days prior to kidney surgery. WT mouse kidneys were subjected to 26 minutes of subthreshold ischemia fol-
lowed by 24 hours of reperfusion. (C) Plasma creatinine was measured 24 hours after sham operation or IRI. Values are mean ± SEM. n = 5–15.
**P < 0.001. (D) Representative morphology (by H&E staining) of kidney outer medulla 24 hours after sham operation or IRI. Scale bar: 100 μm.
Original magnification, ×2 (insets).
4 The Journal of Clinical Investigation http://www.jci.org
sufficient time for an NKT cell response. Compared with mice that
received WT DCs-αGC, administration of WT DC-αGC-ATLs sig-
nificantly protected mouse kidneys from subthreshold IRI, with
lower plasma creatinine levels (Figure 2C) and reduced proximal
tubular cell necrosis, as revealed by H&E staining (Figure 2D).
Similarly, DCs-αGC from Adora2a–/– mice (Adora2a–/– DCs-αGC)
also exacerbated mild kidney IRI compared with control, but
ATL313-treated Adora2a–/– DCs-αGC (Adora2a–/– DCs-αGC-
ATL313) had no protective effect (Figure 2C), thus demonstrating
the selective effect of ATL313. Reduced neutrophil infiltration, as
indicated by FACS analysis, was also observed in mice that were
protected from injury (Table 1 and Supplemental Figure 2).
To examine the duration of efficacy of ATL313-treated DCs, par-
ticularly with regard to future clinical application, we adoptively
transferred either WT or Adora2a–/– DCs-αGC-ATL313 to naive
mice 7 days prior to kidney surgery. Remarkably, the protective
effect of adoptively transferred DCs-αGC-ATL313 lasted for 1 week.
Plasma creatinine levels were approximately 80% lower in mice
that received DCs-αGC-ATL313 1 week prior to kidney IR com-
pared with DC-αGC control (0.35 ± 0.07 vs. 2.01 ± 0.04 mg/dl,
n = 3–4, P < 0.001). This protective effect was not found in
Adora2a–/– DC-αGC-ATL313–treated mice (P > 0.05). These results
suggest that ex vivo treatment of DCs with ATL313 results in a
long-lasting change in vivo either in DC function through activa-
tion of A2ARs, which could be similar to the memory effect of A2AR
signaling described in T cells (30), or in DC target cells.
The subthreshold injury experiments, in which DC-mediated
NKT cell activation is revealed by exacerbation of mild ischemic
injury, were designed to specifically target the protective effect of
DCs-αGC-ATL313 to injury driven by mechanisms involving the
DC-NKT cell-activation pathway. In the next experiments, WT
DCs-αGC-ATL313 were adoptively transferred to WT mice prior
to moderate kidney IR to demonstrate that DCs-αGC-ATL313
can provide protection from a longer ischemic period (28 minute
ischemia/24 hour reperfusion) that alone was sufficient to pro-
duce substantial injury. In contrast to the subthreshold paradigm,
the longer period of ischemia produced maximal injury; IRI alone
produced a large rise in plasma creatinine that could not be fur-
ther increased by administration of DCs or DCs-αGC. Kidneys of
mice that received WT DCs-αGC-ATL313 (but not Adora2a–/– DCs-
αGC-ATL313; NS) were significantly protected from moderate
IRI compared with control mice that received either DCs-αGC or
untreated DCs (Figure 3A). Therefore, we conclude that WT DCs-
αGC-ATL313 are indeed ATL313-induced tolerogenic DCs.
Our studies on immune mechanisms in AKI have employed a
bilateral kidney IRI model involving clamping and release of both
kidney pedicles (6, 16, 26). In recent years, an AKI model was estab-
lished that more closely mimics human renal artery hypoperfusion
during cardiac surgery (35–38). To demonstrate that our results
can be recapitulated in the artery occlusion model, we success-
fully established a mouse renal artery IRI model. DCs-αGC or
DCs-αGC-ATL313 were adoptively transferred to naive WT mice,
and 2 days later, the mice were subjected to renal artery IRI sur-
gery followed by 24-hour reperfusion. We found that DCs-αGC-
ATL313 were also protective in this model; the increase in plasma
creatinine (Figure 3B) and kidney tubule cell necrosis (as shown
by morphology; Supplemental Figure 3A) following renal artery
occlusion was reduced in mice pretreated with DCs-αGC-ATL313.
Administration of DCs-αGC-ATL313 1 or 6 hours after moderate kid-
ney IRI protected kidneys from injury. Our results showing a lasting
effect of DCs-αGC-ATL313 could be applicable in a clinical set-
ting for pretreatment of patients to prevent AKI (e.g., in cardiac
surgery); however, just as important is the need for treatment of
established AKI. We found that kidneys were still protected if mice
received WT DCs-αGC-ATL313 1 or 6 hours after moderate isch-
emia (Figure 3C). Protection was not sustained if adoptive transfer
of DCs-αGC-ATL313 was delayed to 24 hours after the IRI surgery
(Supplemental Figure 3B).
Together, these data provide strong support for the concept that
cell-based therapy with DCs-αGC whose function has been modu-
lated with ATL313 ex vivo can regulate NKT cell activation in vivo
for a period of at least 1 week to protect kidneys from IRI, which
also may be relevant to the control of prolonged graft rejection in
organ transplant, and can be delayed for up to 6 hours following
kidney IR. These findings provide a substantial prospect for clini-
cally relevant AKI or kidney transplant treatment.
DCs-αGC-ATL313 reduce NKT cell responses in vivo and in vitro. Our
previous findings that activation of IL-12/IFN-γ and IL-23/IL-17
pathways promotes neutrophil infiltration in kidney IRI and
NKT cell–mediated inflammation and that IL-17A production is
upstream of and stimulates IFN-γ production by NKT cells and
neutrophils (33) prompted us to determine whether altered plas-
ma IFN-γ and IL-17A/F levels contribute to the protective effects
of ATL313-treated DCs. DC-αGC-ATL313 administration prior
to surgery prevented the increase in IFN-γ and IL-17A/F produc-
tion by DC-αGC–activated NKT cells in WT mice, while Adora2a–/–
DCs-αGC-ATL313 failed to suppress IFN-γ or IL-17A/F produc-
tion (Figure 4, A and B). IL-4 and IL-10 could not be detected in
plasma of DC-αGC-ATL313–treated mice (data not shown), indi-
cating that DC-αGC-ATL313–mediated kidney protection from
IRI does not occur through skewing of Th1- to Th2-type cytokines.
We further examined the mechanisms involved in the suppressive
effect of DCs-αGC-ATL313 in cocultures of DCs and NKT cells.
Coculture with WT DCs-αGC produced a dramatic increase in WT
NKT cell IFN-γ production; however, DCs-αGC-ATL313 failed to
induce NKT cell IFN-γ production in the cocultures (Figure 4C).
Neutrophil numbers in kidneys from mice that received WT or Adora2a–/– DCs ± αGC ± ATL313
WT WT Adora2a–/–
3.44 ± 1.01 (3)
14.51 ± 1.98 (3)
2.13 ± 0.37 (4)
68.58 ± 15.87 (6)A
5.00 ± 0.23 (3)
93.59 ± 35.42 (4)A
2.81 ± 0.83 (3)
74.31 ± 27.76 (5)A
2.37 ± 0.75 (3)
18.56 ± 1.41 (5)
1.80 ± 0.29 (4)
8.61 ± 2.59 (3)
Values (mean ± SEM) are neutrophil number (CD11b+ Gr-1+ cells) in kidney (×104/g kidney) from mice exposed to sham operation or IRI. Numbers in paren-
theses indicate mice per experiment (n). AP < 0.01 compared with WT DCs-αGC-ATL313 IRI.
The Journal of Clinical Investigation http://www.jci.org
Similar results on cytokine production were found when αGC-
loaded WT DCs or αGC-loaded B cells (A20) were cocultured with
NKT hybridoma cells (DN3A4-1.2). Cocultures of NKT hybridoma
cells with WT DCs-αGC-ATL313 (56.3% ± 25.7% reduction, n = 3,
P < 0.05), but not Adora2a–/– DCs-αGC-ATL313 (4.6% ± 7.1% reduc-
tion, n = 3, NS), showed decreased IL-2 production compared with
cocultures with WT DCs-αGC or Adora2a–/– DCs-αGC, respectively.
DCs-αGC-ATL313 do not induce NKT cell anergy. Protection of kid-
neys from IRI by ATL313-treated DCs, including the observed
reduction in cytokine production, could be due to NKT cell
hyporesponsiveness to antigen stimulation. To test whether DCs-
αGC-ATL313 mediated NKT cell anergy, we restimulated isolated
splenocytes from WT mice treated with WT DCs-αGC or DCs-
αGC-ATL313. A comparable increase in IFN-γ production in
response to αGC restimulation was observed in splenocytes iso-
lated from mice treated either with WT DCs-αGC or DCs-αGC-
ATL313 (3.69 ± 3.97 vs. 5.32 ± 2.72, fold of control unstimulated,
NS). These data indicate that DC-αGC-ATL313–mediated kidney
protection does not occur through NKT cell anergy in kidney IRI.
ATL313 affects DC positive and negative costimulatory molecule expres-
sion but not CD1d/glycolipid presentation. Reduced DC-mediated stim-
ulation of NKT cells could reside with altered ATL313-mediated
DC function. We investigated differences in cell phenotype between
DCs-αGC and DCs-αGC-ATL313 by measuring the percentage of
DCs expressing positive (CD80, CD86, CD40, OX40L) and negative
(ICOS, B7-H1, and B7-DC) costimulatory molecules and antigen-
presenting molecules (CD1d, class II [IA]) by flow cytometry. Spe-
cifically, the upregulation of CD40 and OX40L expression that was
found in WT DCs-αGC, compared with WT DCs, was significantly
reduced in WT DCs-αGC-ATL313; ATL313 was ineffective in sup-
pressing CD40 expression in Adora2a–/– DCs-αGC (Figure 5A).
Interestingly, we found that ATL313 upregulated negative costim-
ulatory molecule B7-DC expressed on DCs-αGC (Figure 5A). WT
DCs-αGC-ATL313 had no effect on CD80, CD86 (not shown),
CD1d, IA, ICOS, and B7-H1 expression (Supplemental Figure 4).
To examine whether the suppressive effect of ATL313 on DC-
mediated NKT cell activation might be due to alterations in cellular
processing and antigen presentation of the CD1d/glycolipid com-
plex, we used an antibody (L363) that recognizes CD1d/glycolipid
complex intracellularly and on the cell surface, indicative of antigen
presentation (39, 40). Labeling with antibody L363, as revealed by
FACS, increased significantly both intracellularly (data not shown)
and on the surface of WT or Adora2a–/– DCs after priming with
αGC but was not altered by ATL313 treatment of WT DCs-αGC
(Figure 5B) or Adora2a–/– DCs-αGC (not shown). Furthermore, we
used a B cell line transfected with the murine CD1d molecule (A20.
mCD1d) to investigate αGC trafficking in antigen-presenting cells.
Similar to the BMDCs, αGC loading increased L363 expression in
A20.mCD1d cells and ATL313 had no suppressive effect on sur-
face or intracellular expression (data not shown). The data suggest
that ATL313 does not affect CD1d/glycolipid complex trafficking
inside the cell or presentation on the cell surface.
DC-αGC-ATL313–mediated kidney protection is due to IL-10. Most
recent studies in other models have shown that Tregs play an impor-
tant role in DC-mediated tolerance by secreting IL-10. To explore the
contribution of Tregs to the protective effect of ATL313-treated DCs,
we first measured CD4+CD25+FOXP3+ Tregs by using FACS and
found that there was no difference in numbers of Tregs in kidney and
spleen from WT or Adora2a–/– mice pretreated with WT DCs, DCs-
DCs-αGC-ATL313 administered before or after ischemia protect kid-
neys from moderate IRI. (A) The experimental process was similar to
that in Figure 2, except that WT mice were subjected to 28 minutes of
moderate ischemia and 24 of hours reperfusion. Plasma creatinine was
measured 24 hours after sham operation or IRI. n = 2–4. (B) The exper-
iment was similar to that in A, except that mice were subjected to renal
artery clamping (instead of renal pedicle clamping) for 35 minutes;
plasma creatinine was measured after 24 hours of reperfusion. n = 4
and 5 for DCs-αGC and DCs-αGC-ATL313, respectively. (C) DCs-αGC-
ATL313 were administered 1 or 6 hours after moderate (28 minutes)
kidney ischemia, and plasma creatinine was measured after 24 or 48 hours
of reperfusion (from the end of the ischemic period). n = 3–5. *P < 0.05;
**P < 0.01; ***P < 0.001. Values are mean ± SEM.
6 The Journal of Clinical Investigation http://www.jci.org
αGC, or DCs-αGC-ATL313 alone or prior to kidney ischemia and
24 hours of reperfusion (data not shown). No change in kidney Foxp3
mRNA level was detected in mice that received DCs-αGC-ATL313
compared with DCs-αGC (data not shown). However, kidney Il10
mRNA expression increased 20 hours after pretreatment with
DCs-αGC-ATL313 compared with the DC-αGC group (Figure 6A).
To determine a causal relation between IL-10 and kidney protection
induced by DCs-αGC-ATL313, we injected a blocking antibody
to IL-10 (i.v.) simultaneously with DC adoptive transfer. Blocking
endogenous IL-10 with anti–IL-10 mAbs significantly reversed the
protective effect of DCs-αGC-ATL313 in kidney IRI (Figure 6B).
In a variety of models, increased IL-10 release from tolerogenic
DCs promotes tolerogenic responses and protective function, e.g.,
suppressing autoimmune processes or prolonging graft function
(41). To determine whether DC IL-10 production contributes to
kidney protection, Il10–/– DCs-αGC and Il10–/– DCs-αGC-ATL313
were adoptively transferred to WT mice. Like WT DC-αGC, Il10–/–
DCs-αGC exacerbated mild kidney injury and Il10–/– DCs-αGC-
ATL313 completely reversed mild kidney injury compared with Il10–/–
DCs-αGC (Figure 6C). Administration of WT DCs-αGC-ATL313
to Il10–/– mice did not prevent the increase in creatinine after IRI
(Figure 6D), further confirming that IL-10 is necessary for the pro-
tective effect of DCs-αGC-ATL313 and indicating that the source is
systemic rather than locally produced by the injected DCs.
Spleen cells may be a source of IL-10 in DC-αGC-ATL313–mediated
attenuation of kidney IRI. To look for a systemic source of IL-10 in
DC-αGC-ATL313–mediated kidney protection, we used IL-10 GFP
(X-Vert) reporter mice. Following ischemia and 24 hours of kid-
ney reperfusion in DC-αGC-ATL313–treated mice, we found many
IL-10–producing splenocytes by flow cytometry, and the majority
were CD11c+ DCs and B220+ B cells (Supplemental Figure 5A), but
CD4+ and CD8+ T cells were not IL-10–GFP+ (data not shown).
Interestingly, CD11c+ DCs and B220+ B cells from DC-αGC-
ATL313–treated mice produced more IL-10 based on MFI of
IL-10 (Supplemental Figure 5B). The phenotype of kidney IL-10–
producing cells and the signal mediating IL-10 production from
spleen from CD11c+ DCs and B220+ B cells in DC-αGC-ATL313–
induced protection from IRI will require further study. We also
noted that some IL-10–GFP+ cells, although few, were detected by
immunostaining in kidney outer medulla from mice that received
either PBS or DCs-αGC-ATL313 (data not shown). The signifi-
cance of these cells is not known and will require further study.
DCs-αGC-ATL1223 do not alter FOXP3 or surface PD-1 expression
on Tregs. Although Treg numbers did not increase in DC-αGC-
ATL313–treated mice (data not shown), it is possible that altered
Treg function could contribute to kidney protection. To determine
whether A2AR agonist–treated DCs-αGC reduced kidney injury by
enhancing Treg function, we first determined whether coculture
with ATL-treated DCs-αGC enhanced FOXP3 expression in freshly
isolated Tregs; another A2AR agonist, ATL1223, which has proper-
ties similar to ATL313 and antiinflammatory effects in lung trans-
plantation (22), was used for these experiments. No difference in the
mean expression level of FOXP3, the proportion of Tregs that were
FOXP3 positive at the end of coculture, or expression of surface
programmed death 1 (PD-1) (a member of a family of T cell regula-
tors that suppress immune system response) was observed between
Tregs incubated with untreated DCs, ATL1223-treated DCs, DCs-
αGC, or DCs-αGC-ATL1223 (data not shown). We also determined
the effect of DC coculture on FoxP3 induction in naive CD4+ T
cells (CD4+CD25–) and found no difference among untreated DCs,
ATL1223-treated DCs, DCs-αGC, or DCs-αGC-ATL1223 in their
ability to induce FOXP3 in non-Tregs (data not shown).
Prompted by our findings that A2AR agonist ATL313 reduced kid-
ney IRI through direct effects on DCs in vivo, we demonstrated in
this study that transient treatment of DCs ex vivo with ATL313
during αGC loading resulted in a tolerogenic DC phenotype that
modulated DC-mediated NKT cell activation in vivo and mark-
edly protected kidneys from IRI produced by renal pedicle or renal
artery clamping. Our data showed that ATL313 induced a change
in the BMDC phenotype during DC priming with αGC, a glycolip-
id presented by DCs to activate NKT cells. ATL313 downregulated
positive costimulatory molecule expression (CD40 and OX40L)
and upregulated negative costimulatory molecule B7-DC expres-
sion on DCs, but had no effect on DC antigen presentation (sur-
face and intracellular CD1d/αGC complexes). Adoptive transfer of
DCs-αGC-ATL313 substantially inhibited kidney inflammation
following IRI with less NKT cell activation, IFN-γ, and IL-17A/F
production and neutrophil infiltration. DCs-αGC-ATL313 did
Adoptive transfer of DCs-αGC-ATL313 reduces NKT cell activation. (A and B) WT or Adora2a–/– DCs, DCs-αGC, or DCs-αGC-ATL313 were
adoptively transferred to WT mice, and plasma was collected 18 hours later. Plasma IFN-γ (A) and IL-17A/F (B) levels were measured by ELISA.
n = 3–6. (C) WT or Adora2a–/– DCs, DCs-αGC, or DCs-αGC-ATL313 (primed and incubated for 2.5 days with vehicle or αGC ± ATL313) were
cocultured with WT liver NKT cells for 5 days; then supernatant was collected and IFN-γ level was measured by ELISA. n = 3–4. *P < 0.05;
**P < 0.01. Values are mean ± SEM.
The Journal of Clinical Investigation http://www.jci.org
not switch NKT cell Th1-type cytokine (IFN-γ) production to
Th2-type (IL-4) or cause NKT cell anergy, but DCs-αGC-ATL313
showed reduced secretion of proinflammatory cytokines IL-6
and IL-12p40 (data not shown). DC-αGC-ATL313–treated mice
had enhanced kidney IL-10 mRNA levels, and systemic IL-10 was
important for protection from IRI injury, although Treg numbers
did not change in vivo. We found IL-10–producing cells in spleen
(CD11c+ DC and B220+ B cells) and kidney. To our knowledge, this
is the first study to show the significance of using an A2AR agonist
in tolerizing DCs to block the innate immunity of AKI. Important-
ly, this protective effect persisted for 1 week after administration
of DCs -αGC-ATL313 and could be seen if treatment was delayed
until 6 hours after kidney IRI, thus demonstrating clinical rele-
vance for the use of this approach for established AKI. This study
provides a proof of concept of a potentially new immunotherapeu-
tic strategy for the prevention of tissue injury and graft rejection.
AKI is caused by a variety of conditions and has serious conse-
quences. There is a marked increase in the incidence of AKI and
unacceptably high morbidity and mortality in hospitalized patients
from the past 15 years, and there is an urgent need for effective ther-
apy (42). Numerous factors contribute to the development of AKI.
IRI involves a complex cascade of events, including oxidative stress,
inflammation, and interactions among many cell types (43, 44).
The proinflammatory responses of renal endothelial cells and infil-
trated leukocytes reduce renal blood flow through vascular conges-
tion and promote kidney proximal tubule cell injury.
DCs are a heterogeneous group of cells important in immuni-
ty or tolerance, and the idea of using tolerized DCs in cell-based
therapy of cancer, autoimmune disease, and transplantation has
been under investigation for the past 2 decades (45). However,
most studies have focused on the induction of T cell–tolerogenic
responses. Immune regulation of innate immune response via
tolerogenic DCs is critically important in bridging innate and
adaptive immunity and provides the foundation for use in trans-
plant tolerance of allograft injury (46). Extending this concept, we
believe that designing protocols to tolerize DCs may be useful in
the prevention and even early treatment of AKI (47).
One strategy to block immune response to IRI is to adoptively
transfer tolerized DCs to modulate NKT activation and thereby
limit resident DC function. We and others have shown that NKT
cells contribute to organ IRI (5, 6, 48, 49). Interrupting the DC-NKT
cell interaction, depleting CD1d-restricted NKT cells, or using
NKT cell–deficient mice (CD1d KO) protected kidney from IRI
(6). We also found IFN-γ produced from NKT cells initially acti-
vates the immune response and mediates neutrophil migration
into the inflamed kidney following kidney IRI. Neutrophils are
the major IL-17–producing cells in kidney IRI; IL-17 upregulates
cytokine and chemokine production in kidney IRI (33). Targeting
of NKT cells should be an efficient pathway to blocking the initial
immune response to protect the kidney from IRI.
Adenosine is a nucleoside, locally released in response to cellular
stress, such as IRI and inflammation, which allows tissues to adapt to
hypoxia. Hypoxic conditions can lead to inflammation and vice versa
in a variety of disorders (50). Large amounts of adenosine are released
by injured cells in a hypoxic environment, and HIFs are activated by
hypoxia. Hypoxia, and specifically HIF, increases extracellular con-
centrations of adenosine by stimulating enzymatic conversion from
ATP and AMP and by reducing its cellular uptake. Acting through
multiple receptors, adenosine has long been known to have antiin-
flammatory and tissue-protective effects (21). In a series of studies,
we showed that A2AR agonists protect kidney from IRI (26, 27, 51).
These findings were extended in the current study, confirming that
Changes in costimulatory molecule (CD40, OX40L, and B7-DC) but
not CD1d/glycolipid complex expression on DCs-αGC-ATL313. (A) WT
or Adora2a–/– DCs were primed with vehicle (DC) or αGC in the pres-
ence (αGC-ATL313) or absence (αGC) of ATL313 (1 nM), incubated
for 2.5 days, and analyzed by FACS. Representative flow cytometry his-
togram of CD40, OX40L, and B7-DC from gated CD11c+ BMDCs. (B)
Alexa Fluor 647–labeled L363 was used to detect surface CD1d/αGC
complex by flow cytometry. Representative flow cytometry histogram
of L363 from gated CD11c+ BMDCs. Histograms are representative of
three to four experiments.
8 The Journal of Clinical Investigation http://www.jci.org
DCs are necessary for kidney injury after IR and showing that endog-
enous adenosine acts at DC A2ARs to protect kidneys from injury and
that the protective effect of systemic administration of the A2AR ago-
nist ATL313 is dependent on DC A2ARs. It is possible that the protec-
tive effects of adenosine acting on kidney DCs could be modulated
by changes in receptor expression; however, we found by RT-PCR
that A2AR, A2BR, A1R, and A3R mRNA expression on kidney CD11c+
DCs did not change after IRI. Induction of AR expression may have
occurred at an earlier time point in initiation of inflammation fol-
lowing kidney reperfusion that was no longer evident at 24 hours.
Taken together, these observations led us to explore a new therapeu-
tic strategy to generate tolerized DCs ex vivo and adoptively transfer
them to mice to block the innate immune system and prevent kid-
ney injury following IRI. More specifically, our strategy was to sup-
press NKT cell activation by using NKT cell–specific antigen–loaded
(αGC) DCs treated with an A2AR agonist ex vivo.
Adenosine may act on other ARs to mediate protection. Recent-
ly, Grenz et al. demonstrated an important homeostatic role of
equilibrative nucleoside transporter 1 (ENT1); they showed that
ENT1 regulates uptake of adenosine and that crosstalk between
renal ENTs and vascular endothelial A2BR protected kidneys from
ischemic injury by improving reperfusion (52). In the current
study, we extended our previous findings, that A2AR expressed
on BM-derived cells contributes to suppression of kidney IRI, by
demonstrating a critical role for A2AR expressed on CD11c+ DCs
both in kidney IRI and as a target for ex vivo modulation of DC
function to block DC-mediated NKT cell activation in kidney IRI.
By reducing leukocyte infiltration and preventing increases in
adhesion molecule expression (53), A2AR stimulation also reduces
vascular congestion, thus improving renal blood flow by mecha-
nisms that may complement the effects described by Grenz et al.
(52). Thus, by broadly acting at multiple cellular sites to restore
IL-10 plays a role in DC-αGC-ATL313–mediated protection from kidney IRI. (A) Kidney RNA was isolated from naive mice 20 hours after adoptive
transfer of WT DCs, DCs-αGC, or DCs-αGC-ATL313, and Il10 mRNA levels were measured by real-time PCR. Values are fold change relative to
DCs. n = 3. (B) Two days before mild IRI, neutralization IL-10 mAb or IgG2a isotype control (200 μg, i.p) was administered to mice during pretreat-
ment with DCs-αGC or DCs-αGC-ATL313. n = 4–5. (C) αGC-loaded Il10–/– DCs treated either with vehicle or ATL313 (1 nM) or WT DCs-αGC were
adoptively transferred to WT mice 2 days prior to mild (26 minutes) kidney ischemia. n = 3–9. (D) PBS, WT DCs-αGC, or WT DCs-αGC-ATL313
were administered to Il10–/– mice 2 days prior to moderate IRI. n = 3 for DCs-αGC or DCs-αGC-ATL313. Plasma creatinine (B–D) was measured
following 24 hours of kidney reperfusion. *P < 0.05; ***P < 0.001. Values are mean ± SEM.
The Journal of Clinical Investigation http://www.jci.org
function and dampen inflammation, adenosine’s action at differ-
ent AR subtypes on distinct hematopoietic and nonhematopoietic
cells could provide powerful nonredundant (additive or synergis-
tic) homeostatic tissue protection.
Compared with mature DCs, immature DCs interact actively with
T cells and direct them into a regulatory response. There are several
strategies used to maintain the immature state of DCs and gener-
ate tolerogenic DCs (1). These include culturing DCs with different
cytokines (e.g., IL-10, TGF-β), growth factors (GM-CSF), or immu-
nosuppressive agents (e.g., corticosteroids, vitamin D3, cyclosporine,
tacrolimus, rapamycin), modulation of the expression of costimula-
tory molecules, and genetic interference. Tolerized DCs may sup-
press immune responses through various mechanisms. Tolerance is
influenced by suppressing positive costimulatory molecules (CD80,
CD86, and CD40), upregulating negative costimulatory molecules
(PD-1, B7-H1, and ICOS) (54), and inhibiting cytokines, such as
IL-12p70 and IL-12p40 (55). The IL-12 family member IL-27 (com-
prising EBI3 and IL-27–p28) is critical for tolerized DCs to produce
the antiinflammatory cytokine IL-10 (56). Epstein-Barr virus–
induced gene 3 (EBI3) pairs with IL-12p35 to form IL-35, which is a
mediator for Treg differentiation and is important in maintaining
an immunosuppressive state (57). Finally, TGF-β, retinoic acid, and
vitamin D3 appear to be important in peripheral tolerance through
Treg differentiation and expansion (58).
Although we did not find that DCs-αGC-ATL313 promoted kid-
ney or spleen CD4+CD25+FOXP3+ Treg numbers, we found that
systemic IL-10 played an important role in suppression of kidney
IRI (58). IL-10 and heme oxygenase 1 are important in prolong-
ing skin allograft survival (59). Precise determinations of the phe-
notype and function of cells that contribute to systemic IL-10 will
require additional study; however, IL-10–producing cells (CD11c+
DCs and B220+ B cells) were detected in spleen. Kidney IRI has been
recognized as a systemic inflammatory process, and splenectomy
ameliorates acute multiple organ damage in IR models (60). The
role of IL-10 production from CD11c+ DCs and B220+ B cells in
tissue protection mediated by ATL313-induced tolerogenic DCs
will require further studies. A subset of IL-10–producing human
DCs, termed DC-10, are present in vivo and are potent inducers of
antigen-specific IL-10–producing Tregs (61). IL-10–differentiated
DCs (DC-10) induce tolerance at least in part by inducing T effector
cells to differentiate into CD4+CD25hiFOXP3+ Tregs (62). The defec-
tive generation of IL-10–induced tolerogenic DCs and iTregs may
contribute to inflammatory changes in hyper-IgE syndrome (63).
Exogenously administered tolerogenic DCs may change the pheno-
type of the kidney resident DCs and/or further promote tolerance
by inducing resident DCs to become IL-10–producing tolerogenic
DCs, as we observed increased IL-10 mRNA expression in DC-αGC-
ATL313–treated naive WT kidneys. To examine the possibility that
non–BM-derived cells can produce IL-10 to mediate tolerogenic
DC-induced protection from kidney IRI, future studies using BM
chimera will be needed. DCs and marginal zone B cells may coordi-
nate together to initiate T/NKT cell responses. IL-10 produced from
B220+ cells contributed to early T cell regulation (64, 65). Although
increased systemic levels of Th2 cytokines IL-4 and IL-10 could not
be detected in DC-αGC-ATL313–treated mice, local tissue levels of
these antiinflammatory cytokines may mediate protection from
kidney IRI following infusion of ATL313-induced tolerogenic DCs.
Recent studies have demonstrated that Tregs have the ability to
protect the kidney from ischemic injury and inflammation (66,
67), and our findings on IL-10 would suggest that Tregs may be
involved in the protective effect of tolerogenic DCs. PD-1 is a
member of a family of T cell regulators that suppress immune
system responses. Treg-mediated protection can be overcome by
blocking PD-1 on the surface of Tregs, suggesting that Treg sur-
face PD-1 expression is vital for their action in this model. Fur-
thermore, PD-1–KO Tregs have reduced ability to suppress both
CD4+ and CD8+ T cell activation (68). We hypothesized that DCs-
αGC-ATL313 could be protecting the kidney by enhancing the
functional properties of Tregs. However, DCs-αGC-ATL1223 had
no effect on FOXP3 expression in Tregs or non-Treg CD4+ T cells,
and A2AR agonist–treated DCs (in the presence or absence of αGC)
had no effect on the expression of PD-1 on the surface of Tregs.
There are many challenges in using the approach of tolerized
DCs in blocking the innate immune system, e.g., standardizing
treatment strategies, as DCs represent a heterogeneous popula-
tion that express different surface markers in different tissues. Our
study showed that at least a partial protective effect of ATL313-
induced tolerized DCs was sustained for 1 week after adoptive
transfer, which could be relevant for some clinical indications,
such as prophylaxis of AKI in high-risk patients. In addition, the
route of delivery of tolerized DCs may affect tolerized DC hom-
ing efficiency and their immune regulatory function. We found
most of the adoptively transferred DCs migrated to the lymphoid
nodes, spleen, and other organs, including kidney (our unpub-
lished observations). Other DC subtypes, such as plasmacytoid
DCs, also should be considered for therapeutic use.
In summary, through step-wise ex vivo DC manipulation and
administration in an in vivo kidney IRI model, our study has iden-
tified a cell-based strategy to prevent AKI and potentially allograft
rejection. We have demonstrated that A2AR agonist ATL313-
induced DC tolerance shows strong biological activity to prevent
NKT cell–mediated innate immune activation in vitro and in vivo
and further regulate immunosuppression in kidney IRI.
Mice and surgical protocol. WT C57BL/6 mice (6 to 8 weeks old; NCI) were
used, and C57BL/6 background Il10–/–, Ifng–/–, CD11c Cre, and IL-10–IRES-
eGFP reporter mice (Vert-X) were purchased from The Jackson Laboratory.
Adora2a–/– mice (C57BL/6 background, 6 to 7 weeks of age), generated as
previously described (69), were a gift from J.-F. Chen (Boston University,
Boston, Massachusetts, USA). KO mice were backcrossed with C57BL/6
background mice for at least 8 generations.
Mice were anesthetized with a mixture (i.p.) of ketamine (120 mg/kg),
xylazine (12 mg/kg), and atropine (0.324 mg/kg) and were subjected to
bilateral flank incisions. Both kidney pedicles were exposed and cross-
clamped for 26 minutes (subthreshold) to produce mild injury (in order
to evaluate exacerbation of injury) or in some experiments for 28 minutes
to produce moderate injury; then clamps were released (reperfusion) for
24 hours as described before (25). Kidney pedicles were exposed but not
clamped in sham-operated mice. During the surgery, mouse core tempera-
ture was maintained at 34–36°C with a heating pad; during the recovery
and reperfusion period, mice were housed in a warming incubator with
ambient temperature at 30–32°C.
For renal artery IRI, mice were subjected to bilateral flank incisions. On
the right side, surgical thread was tied around the renal vessels, a cut was
made above the suture to extract the kidney, and the incision was closed.
The ureter and kidney blood vessels were secured with minimal blood loss.
Kidney artery and vein were dissected under a Zeiss Stemi 2000-C Stereo
Microscope (Carl Zeiss Microscopy LLC). The kidney artery was clamped
for a period of 35 minutes by using a clip with a pressure of 70 g (Roboz
10 The Journal of Clinical Investigation http://www.jci.org
Surgical Instrument); then the clip was released to allow the kidney to
reperfuse. Control of temperature during surgery and reperfusion was the
same as in the bilateral clamping procedure.
Following 24 hours of reperfusion, animals were reanesthetized, blood
was obtained from the right retroorbital sinus, and kidneys were removed
for various analyses. Plasma creatinine as a measure of kidney function
was determined using a colorimetric assay according to the manufacturer’s
The optimal dose of neutralization antibodies (200 μg/mouse for
anti–IL-10; BioXCell) or their respective isotype control (IgG2a) was
administrated i.p. at the onset of BMDC adoptive transfer 2 days prior
to kidney surgery. Osmotic mini-pumps (Alzet Osmotic Pumps) pre-
loaded with sterile saline vehicle, ATL146e (at a concentration calcu-
lated to deliver 10 ng/kg/min; Adenosine Therapeutics LLC), or ATL313
(1 ng/kg/min, Adenosine Therapeutics LLC) were introduced s.c. 24 hours
prior to kidney surgery or at the onset of BMDC adoptive transfer,
depending on the experimental design.
Generation of CD11c-Cre Adora2afl/WT and Adora2afl/fl mice. The Cre-loxP
strategy was used to generate a CD11c+ DC–specific deletion of A2AR
in mice. Briefly, homozygous Adora2afl/fl mice (C57BL/6 background;
Adora2a gene modified by floxed sequences flanking exon 2) (32) were
bred with CD11c-Cre transgenic mice (The Jackson Laboratory), express-
ing Cre recombinase under the control of the CD11c promoter, to gener-
ate CD11c-CreAdora2afl/WT and CD11c-CreAdora2afl/fl mice that lack A2ARs
in DCs. CD11c-CreAdora2afl/WT, CD11c-CreAdora2afl/fl, and control mice
(CD11c-Cre, Adora2afl/fl, and Adora2afl/WT) were genotyped using DNA
from tail biopsies with the following primer sets: (a) for CD11c-Cre:
Cre forward, 5′-AGGTGTAGAGAAGGCACTTAG, and Cre reverse,
5′-CTAATCGCCATCTTCCAGCAGG, which generated a 411-bp prod-
uct; and (b) for Adora2a WT (Adora2aWT) and floxed Adora2a alleles:
forward 5′-GGGCAAGATGGGAGTCATT-3′ and reverse 1-5′-ATTCT-
GCATCTCCCGAAACC-3′ and reverse 2-5′-AACAGTTATTCT-
GATCTTTCC -3′, which generated a 216-bp product for Adora2afl/fl and
an 182-bp product for the WT DNA sequence.
Kidney and spleen tissue digestion and FACS analysis of leukocytes. Kidney
or spleen cell suspensions were prepared from mice subjected to IRI or
sham operation, and kidney leukocyte subset cell number was calculated
as described before (6). The following antibodies (1 μg/ml; eBioscience)
were used to identify kidney neutrophils: anti-mouse CD45-APC-Alexa
Fluor 750 (30-F11), CD11b-PE (M1/70), and GR-1-APC (Ly6G RB6-8C5).
7-AAD was used to exclude dead cells. CD45-labeled samples were fur-
ther labeled with CD11c antibodies to identify DCs expressing costimu-
latory markers (with anti-mouse CD80, CD86, CD40, OX40L, ICOS,
B7-H1, and B7-DC) and antigen-presenting molecules (with anti-mouse
CD1d and MHC class II [IA]) (eBioscience), as described previously (6).
CD1d tetramer loaded with PBS-57 (1:100), an analog of α-GC (NIH
Tetramer Core Facility, Emory University, Atlanta, Georgia, USA) (6),
and anti-mouse TCR-β (eBioscience) were used to identify NKT cells.
Intracellular staining for IFN-γ was performed using the BD Biosciences
Fix/Perm buffer set according to the manufacturer’s protocol and as
described previously (6). Tregs were labeled with anti-mouse antibod-
ies (clones) from eBioscience (CD45 [Ly-5] APC-eFluor 780, PD-1 [J43]
PE, CD25 [PC61.5] PE, FOXP3 [FJK-16s] Alexa Fluor 647) and from
BD Biosciences (CD4 [RM4-5] V500) (66). IL-10–producing GFP+ cells
from kidney and spleen of Vert-X mice were identified by gating on the
CD45+7-AAD– live leukocyte population; additional antibodies included
anti-mouse CD11c, CD4, CD8, and B220 (eBioscience). Flow cytometry
data was acquired on BD FACSCalibur (BD Bioscience) with Cytek 8
Color Flow Cytometry Upgrade (Cytek Development Inc.) and analyzed
by FlowJo software 9.0 (Tree Star Inc.).
Quantitative real-time RT-PCR. Total RNA was extracted from kidneys
with TriReagent according to the manufacturer’s protocol (Molecular
Research Center Inc.) and reverse transcribed with cDNA transcript kit
(Invitrogen). Primers were designed using IDT PrimerQuest (Integrated
DNA Technologies; www.idtdna.com). Primer sequences for Il10 and
FoxP3 mRNA are listed in Supplemental Table 1. RT-PCR was performed
using the iScript 1-step RT-PCR kit with SYBR Green (Bio-Rad), and sam-
ples were normalized to RPS29.
Kidney CD11c+ DC isolation and real-time PCR detection of adenosine A1R, A2AR,
A2BR, and A3R receptors. Kidneys from mice exposed to sham operation or
IRI were digested as described above for FACS analysis. Kidney cell suspen-
sions were washed, cells were resuspended in 30% Percoll (GE Healthcare
Life Science), and the samples were gently laid over 70% Percoll and cen-
trifuged at 960 g for 20 minutes at room temperature. Mononuclear cells
were collected, washed, and stained with anti-mouse CD45-PECy7, CD11c-
APC–Alexa Fluor 780 (eBioscience), and 7-AAD. CD45+CD11chi7AAD–
cells were sorted with an i-Cyt Reflection Cell Sorter (UVA Flow Cytom-
etry Core). RNA isolation from sorted kidney DCs was performed by using
QIAGEN RNeasy Mini Kit (QIAGEN). The primers for adenosine A1R,
A2AR, A2BR, and A3R receptors are shown in Supplemental Table 1.
Detection of CD1d/αGC complex with L363 mAbs. BMDCs or A20 and CD1d-
transfected (A20.CD1d) B cell lines (provided by Mitchell Kronenberg, La
Jolla Institute for Allergy and Immunology) were cultured and treated with
vehicle, αGC (0.1 μM), or αGC plus ATL313 (1 nM) for 2.5 days. L363 was
labeled with Alexa Fluor 647 by using the APEX Alexa Fluor 647 Antibody
Labeling Kit (Invitrogen). Cells were collected and Alexa Fluor 647–L363
(10 μg/ml) surface and intracellular staining were performed by using BD
Cytofix/Cytoperm Kit (BD Biosciences). 7-AAD was used to exclude dead
cells. Cells were analyzed by flow cytometry as described above.
Priming BMDCs with αGC. As described before (33), BM cells were isolated
from WT, Adora2a–/–, or Il10–/– mice and >90% pure DCs were obtained fol-
lowing 2 weeks in culture. DCs were primed with vehicle (0.1% DMSO in cul-
ture medium; DC) or 0.1 μg/ml αGC (AXXORA, LLC; DC-αGC) for 2.5 days
with or without 1 nM ATL313. Cells were then washed and 0.5 × 106 cells/
mouse (optimal dose) were adoptively transferred (i.v.) to naive mice 2 days
prior to kidney IR surgery (subthreshold, mild [26 minutes] or moderate
[28 minutes] ischemic injury).
Coculture of DCs with NKT cells in vitro. Naive WT mouse liver tissue was
digested with type I collagenase (10 μg/ml) for 20 minutes and mashed.
Mononuclear cells were isolated over 35% Percoll. 10 μg/ml PE-labeled
anti-CD1d tetramer (NIH Tetramer Core Facility, Emory University) was
added to the liver mononuclear cells to stain NKT cells; then anti-PE micro-
beads were used to isolate NKT cells through MACS columns as described
by the manufacturer (Miltenyi Biotec). 1 × 104 WT DCs, DCs-αGC,
or DCs-αGC-ATL313 were cocultured with 5 × 104 NKT cells in 96-well
round-bottom plates for 5 days, and supernatants were collected for anal-
ysis of IFN-γ by ELISA (eBioscience).
Similarly, 5 × 105/ml DCs, DCs-αGC, or DCs-αGC-ATL313 were cocul-
tured with Vα14Vβ8.2 NKT cell DN3A4-1.2 hybridomas (1.2) in 96-well
round-bottom plates, and supernatant IL-2 was measured by ELISA
(eBioscience) according to published protocols (70). αGC- or αGC-
ATL313–loaded B cells (A20) were also cocultured with NKT cells to test
NKT cell response by measuring IL-2 levels.
NKT cell anergy test. Splenocytes were isolated from WT mice pretreated for
2 days with either WT or Adora2a–/– DCs, DCs-αGC, or DCs-αGC-ATL313.
1 × 106 splenocytes were restimulated with vehicle or αGC (0.1 μg/ml)
in 96-well round-bottom plates for 24 hours, and supernatants were col-
lected for IFN-γ assay by ELISA (eBioscience).
Histochemistry. Kidneys were fixed and processed for H&E staining as
previously described (6) and viewed by light microscopy (Zeiss AxioSkop)
The Journal of Clinical Investigation http://www.jci.org
This work was supported in part by funds from NIH
RO1DK56223, RO1DK62324, P01HL073361, Genzyme (Gen-
zyme Renal Innovations Program), and American Heart Associa-
tion National Scientist Development Grant 0835258N. We thank
Steven A. Porcelli (Albert Einstein College of Medicine, New
York, New York, USA) for generously providing L363 mAbs and
Mitchell Kronenberg (La Jolla Institute for Allergy and Immu-
nology) for providing the Vα14Vβ8.2 DN3A4-1.2 (1.2) NKT cell
hybridomas, A20 B cell line, and transfected murine CD1d A20.
CD1d cell line. We also thank the University of Virginia Research
Histology Core and Flow Cytometry Core Facilities.
Received for publication February 1, 2012, and accepted in revised
form August 16, 2012.
Address correspondence to: Li Li, Division of Nephrology, Box
800746, University of Virginia Health System, Charlottesville,
Virginia 22908, USA. Phone: 434.982.6661; Fax: 434.982.1616;
Sang Ju Lee’s present address is: Division of Nephrology, Depart-
ment of Internal Medicine, Daejeon St. Mary’s Hospital, The
Catholic University of Korea, Daeheungdong, Chungku, Daejeon,
301-723, Republic of Korea.
Emily K. Moser’s present address is: Department of Pharmacology,
Beirne B. Carter Center for Immunology Research, University of
Virginia Health System, Charlottesville, Virginia, USA.
under ×200 magnification. Photographs were taken and brightness/con-
trast adjustment was made with a SPOT RT camera (software version 3.3;
Plasma cytokine detection by ELISA. Plasma was collected from mice 20 hours
after administration of DCs, DCs-αGC, or DCs-αGC-ATL313. Plasma
IFN-γ, IL-17A/F, IL-4, and IL-10 levels were measured by using mouse
ELISA kits (eBioscience) following the manufacturer’s protocol.
DC and Treg coculture. DCs were isolated and cultured as described above
and exposed to 10 nM ATL1223 (Dogwood Pharmaceuticals Inc., a wholly
owned subsidiary of Forest Laboratories Inc. ) and/or 100 ng/ml αGC for
2 days, then washed prior to initiation of cocultures with freshly isolated
Tregs. Tregs were isolated from spleens of naive WT CD45.1 mice using the
Dynal CD4+ T cell–negative isolation kit followed by the CD25–positive iso-
lation kit from Miltenyi Biotec according to the manufacturers’ protocols
and as described previously (71). Cocultures consisted of 5 × 104 DCs plus
1 × 105 Tregs in a total of 250 μl of medium in round-bottom 96-well plates
for 48 hours. No activation stimuli were added (e.g., anti-CD3 or anti-
CD28). Subsequently, FOXP3 staining for flow cytometry was conducted
using the eBioscience FOXP3 Staining Buffer Set according to the manu-
facturer’s protocol (66) The anti–PD-1 (29F.1A12) antibody (Biolegend)
was used only prior to permeabilization of the cells.
Statistics. GraphPad Instat 3 (GraphPad Inc.), SigmaPlot 11.0 (Systat
Software Inc.), and Canvas X (ACD Systems of America Inc.) were used to
analyze and present the data. Data were analyzed, after transformation if
needed to generate a normal distribution, by t test or 2-way ANOVA with
post hoc analysis as appropriate. P < 0.05 was used to indicate significance.
Study approval. All experiments were performed in accordance with NIH
and Institutional Animal Care and Use Guidelines and were approved by
the Animal Care and Use Committee at the University of Virginia.
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