In vivo-mobilized kidney dendritic cells are functionally immature, subvert alloreactive T-cell responses, and prolong organ allograft survival.
ABSTRACT Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L).
Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined.
Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy.
Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.
- SourceAvailable from: nature.com[Show abstract] [Hide abstract]
ABSTRACT: With their ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great potential for the treatment of currently incurable diseases. Two hESC-based cell therapies for spinal cord injury and macular degeneration have been advanced into human clinical trials. Despite this rapid progress, one key challenge of hESC-based cell therapy is the allogeneic immune rejection of hESC-derived cells by recipients. This problem could be mitigated by a recent breakthrough in the technology of induced pluripotent stem cells (iPSCs) by nuclear reprogramming of patient-specific somatic cells with defined factors, which could become a renewable source of autologous cells for cell therapy. However, recent studies revealing the abnormal epigenetics, genomic stability and immunogenicity of iPSCs have raised safety concerns over iPSC-based therapy. Recent findings related to the immunogenicity of iPSC derivatives will be summarized in this review.Cellular & Molecular Immunology advance online publication, 16 December 2013; doi:10.1038/cmi.2013.60.Cellular & molecular immunology 12/2013; · 4.19 Impact Factor
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ABSTRACT: The induction of immune tolerance is still a formidable challenge in organ transplantation. Dendritic cells (DCs) play an important role in orchestrating immune responses by either mediating protective immune responses or inducing antigen specific tolerance. Previous studies demonstrated that the fms-like tyrosine kinase 3 receptor (Flt3) and its ligand (Flt3L) play an essential role in the regulation of DC commitment and development. Here, we report a synergic effect between Flt3L and low-dose rapamycin (Rapa) in the protection of allograft rejction. It was found that Flt3L combined with Rapa significantly prolonged murine cardiac allograft survival time as compared with that of untreated recipients or recipients treated with Rapa or Flt3L alone. Mechanistic studies revealed that Flt3L combined with low-dose of Rapa induced the generation of tolerogenic DCs along with the production of CD25(+) Foxp3(+) regulatory T cells and IL-10 secretion. We also observed enhanced autophagy in the cardiac allograft, which could be another asset contributing to the enhanced allograft survival. All together, these data suggest that Flt3L combined with low-dose of Rapa could be an effective therapeutic approach to induce tolerance in clinical setting of transplantation.PLoS ONE 10/2012; 7(10):e46230. · 3.53 Impact Factor
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Copyright © 2004 by Lippincott Williams & Wilkins, Inc.
Vol. 77, GGG–GGG, No. 7, April 15, 2004
Printed in U.S.A.
IN VIVO-MOBILIZED KIDNEY DENDRITIC CELLS ARE
FUNCTIONALLY IMMATURE, SUBVERT ALLOREACTIVE T-CELL
RESPONSES, AND PROLONG ORGAN ALLOGRAFT SURVIVAL1
P. TOBY H. COATES,2,3F. JASON DUNCAN,2BRIDGET L. COLVIN,2,3ZHILIANG WANG,2
ALAN F. ZAHORCHAK,2WILLIAM J. SHUFESKY,2ADRIAN E. MORELLI,2AND ANGUS W. THOMSON2,4,5
Background. Migratory antigen-presenting cells res-
ident in kidneys may have tolerogenic potential. Dif-
ficulties inherent in their isolation have limited their
characterization. The authors examined the pheno-
type and function of murine kidney dendritic cells
(DC) mobilized in vivo by systemic administration of
fms-like tyrosine 3 kinase ligand (Flt3L).
Methods. Monoclonal antibody staining was used to
characterize DC subsets in situ, immediately after
their isolation, and after lipopolysaccharide stimula-
tion. Cytokine and CC chemokine receptor (CCR) gene
expression was analyzed by RNase protection assay.
Mixed leukocyte reactions were performed to assess
DC allostimulatory ability and also the function of
putative T-regulatory cells. In vivo DC trafficking was
monitored by fluorescence imaging of dye-labeled
cells and the influence of renal DC on vascularized
heart allograft survival was determined.
Results. Flt3L induced a marked increase both in
CD11c?CD8??and in CD11c?CD8??DC within the
renal cortex and medulla. Rarer, CD11c?B220?(pre-
cursor plasmacytoid) DC were also detected. Bulk
freshly isolated DC exhibited no interleukin (IL)-
12p35 mRNA, low surface co-stimulatory molecule ex-
pression, and CCR transcripts, consistent with imma-
turity. They elicited only weak allogeneic T-cell
proliferative responses, and repeated stimulation in-
duced CD4?CD25?IL-10?T cells. In vivo, the freshly
isolated DC failed to prime T cells of naive allogeneic
hosts for anti-donor cytotoxic T-cell responses. When
infused systemically, 1 week before organ transplan-
tation, they prolonged graft survival without immuno-
Conclusions. Hematopoietin-mobilized renal DC are
functionally immature and exhibit tolerogenic poten-
tial. Mobilization of DC within kidneys is likely to
affect their antigen-handling capacity, immunogenic-
ity, and tolerogenic ability.
Dendritic cells (DC) are uniquely well-equipped bone mar-
row-derived antigen (Ag)-presenting cells (APC) distributed
throughout the body that determine the balance between
tolerance and immunity (1–3). In peripheral nonlymphoid
tissues, such as the skin, airways, and intestinal epithelium,
DC are found as immature interstitial APC. In the normal
steady state, these immature DC in the periphery may play
an important role in the maintenance of self-tolerance (4).
After activation, they migrate in increased numbers through
the lymphatics to secondary lymphoid tissue. In the process,
they undergo phenotypic and functional maturation and ac-
quire the ability to stimulate Ag-specific T cells (1). Their
unique capacity to activate naive T cells reflects high surface
levels of major histocompatibility complex (MHC) and co-
stimulatory molecules and the ability to secrete bioactive
interleukin (IL)-12p70. By contrast, immature DC freshly
isolated from nonlymphoid tissues, or propagated in vitro,
lack adequate co-stimulatory molecules and induce Ag-spe-
cific T-cell hyporesponsiveness (5). Immature DC have also
been shown to skew T-helper (Th) cell responses toward Th2
predominance (6), to induce T-regulatory cells (Treg) (7), and
to prolong the survival of organ allografts (8, 9).
Renal DC were first described in rats as Ia?(MHC class
II?) cells in immunohistochemical studies by Hart and Fabre
in 1981 (10) and later implicated as the principal instigators
of kidney allograft rejection (11). Similar interstitial (MHC
class II?) cells have been shown to migrate from the kidney
after administration of bacterial lipopolysaccharide (LPS) to
normal rodents (12). Despite the perceived importance of
renal DC in responses to local infection or tumor develop-
ment, or in the initiation of graft rejection, comparatively
little is known about the functional immunobiology of these
cells. Intriguingly, murine kidney grafts, like liver trans-
plants, may be accepted across MHC barriers (including
H2b3H2k) without immunosuppression (13), whereas in
large animals, simultaneous transplantation of donor kid-
neys induces tolerance to heart allografts (14). These findings
call into question the role of donor renal DC in transplant
tolerance versus rejection. Studies on kidney DC have been
limited because of their paucity in normal tissue and because
of the difficulties inherent in their isolation. Nevertheless, a
decade ago, Austyn et al. (15) described low-buoyant-density
MHC II?cells with little or no T-cell stimulatory activity
when freshly isolated from large numbers of normal mouse
kidneys. More recently, the endogenous hematopoietic
1This work was supported by National Institutes of Health grants
R01 DK 49745 and R01AI 41011 (A.W.T.) and R21 HL69725 and R21
AI55027 (A.E.M.). P. Toby H. Coates is a Don and Lorraine Jacquot
Traveling Fellow, Royal Australian College of Physicians, and the
recipient of a C. J. Martin Fellowship from the National Health and
Medical Research Council of Australia. Bridget L. Colvin is sup-
ported by a National Cancer Institute predoctoral fellowship train-
ing award (P32CA82084).
2Thomas E. Starzl Transplantation Institute and Department of
Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA.
3Thomas E. Starzl Transplantation Institute and Department of
Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA;
currently, Renal Unit, The Queen Elizabeth Hospital, Woodville,
South Australia, Australia.
4Department of Immunology, University of Pittsburgh Medical
Center, Pittsburgh, PA.
5Address correspondence to: Angus W. Thomson, M.D., W1544
Biomedical Science Tower, University of Pittsburgh Medical Center,
200 Lothrop Street, Pittsburgh, PA 15213. Email: thomsonaw@msx.
Received 18 August 2003.
Revision requested 16 September 2003. Accepted 1 December 2003.
balt5/ztr-tp/ztr-tp/ztr00704/ztr7010-04amarksjS?53/1/04 14:194/Color Figure(s): 1,5 Art: 145344
<ARTICLE DOCTOPIC??IMMUNOBIOLOGY? DOCSUBJ??&NA;? DATE??April 15, 2004? VID??77? ISS??7? PPF??GGG? PPL??GGG? DOI??10.1097/01.TP.0000122183.60680.C9?>
growth factor fms-like tyrosine kinase 3 ligand (Flt3L) has
been shown to dramatically increase DC in bone marrow,
blood, and lymphoid organs (16). Its influence on kidney DC
has not been examined. We report in this article that Flt3L
markedly increases interstitial renal DC, facilitating inves-
tigation of their functional immunobiology. Our data show
that DC recruited to the kidney in response to this poietin are
functionally immature and exhibit tolerogenic properties.
These in vivo-mobilized DC have potential to alter the kid-
ney’s Ag-handling ability and its immunogenicity.
MATERIALS AND METHODS
Male C57BL/10J (B10;H2b), C3H/HeJ (C3H;H2k), and BALB/c
mice (BALB/c;H2d), 8 to 12 weeks of age, were purchased from The
Jackson Laboratory (Bar Harbor, ME) and housed in the specific
pathogen-free facility of the University of Pittsburgh Medical Cen-
ter. They were provided with Purina rodent chow (Ralston Purina,
St. Louis, MO) and fresh water ad libitum. Experiments were con-
ducted in accordance with the National Institutes of Health Guide
for the Care and Use of Laboratory Animals and under an institu-
tional animal care and use committee-approved protocol.
Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or cyto-
chrome c-conjugated monoclonal antibodies (mAb) directed against
CD3?, CD8?, CD11b, CD11c, CD40, CD45R/B220, CD80, CD86, and
MHC class II were purchased from BD PharMingen (San Diego, CA).
Chinese hamster ovary cell-derived recombinant human Flt3L was
kindly provided by the Immunex Corporation (now Amgen, Seattle,
WA). RPMI-1640 (Life Technologies, Gaithersburg, MD) was supple-
mented with 10% v/v fetal calf serum (Nalgene, Miami, FL), nones-
sential amino acids, L-glutamine, sodium pyruvate, penicillin-strep-
tomycin, and ?2-mercaptoethanol (all from Life Technologies) and is
referred to subsequently as complete medium. Murine recombinant
granulocyte-macrophage colony-stimulating factor was provided by
Dr. S. K. Narula (Schering-Plough Research Institute, Kenilworth,
NJ). Bacterial (Escherichia coli) LPS was obtained from Sigma (St.
Louis, MO). Phosphate-buffered saline (PBS) without calcium or
magnesium was obtained from BioWhittaker (Walkersville, MD).
DC were detected and characterized in normal and Flt3L-treated
mouse kidneys by immunofluorescence staining for CD11c, CD8?,
and CD86. Tissue blocks were embedded in Tissue-Tek OCT (Miles
Laboratories, Elkhart, NJ), snap-frozen in isopentane (prechilled in
liquid nitrogen), and stored at ?80°C until use. Eight-micron cryo-
stat sections were mounted on slides pretreated with Vectabond
(Vector Laboratories, Burlingame, CA) and then air-dried and fixed
in cold acetone (4°C). The sections were then incubated with normal
goat serum, avidin blocking solution (Vector), PE-conjugated anti-
CD11c, and FITC-conjugated rat anti-mouse CD8? or FITC-conju-
gated hamster anti-mouse CD86. After rinsing, nuclei were counter-
Kidney DC Isolation
DC were isolated from kidneys of normal mice or animals treated
with Flt3L (10 ?g/mouse/day administered intraperitoneally in PBS)
for 10 consecutive days. In a typical experiment, four kidneys were
finely dissected and incubated in 10 mL of complete medium supple-
mented with type IV collagenase (1 mg/mL; Sigma), 1 mL of trypsin
(0.25%; Life Technologies), and DNAase I (0.5 mL of a 1-mg/mL
solution in PBS) in a humidified atmosphere of 5% carbon dioxide in
air at 37°C. After 45 min, the digested tissue was filtered through
fine mesh at room temperature. Nonparenchymal cells in the super-
natants of three sequential spins for 5 min at 40g were pooled and
then centrifuged at 1,000g for 7 min. DC were enriched by density
gradient separation using 42% w/v metrizamide (Sigma) at 1,200g
for 30 min at 4°C. CD11c?cells were then positively selected as
described (17), using immunomagnetic beads (Miltenyi Biotech, Au-
burn, CA) and the Macs separation system. The purity of immuno-
bead-sorted cells was consistently greater than 85%. To promote DC
viability and maturation, CD11c?bead-purified cells were cultured
overnight (106cells/mL) in 24-well plates (Corning, Corning, NY) at
37°C in complete medium supplemented with granulocyte-macrophage
colony-stimulating factor (4 ng/mL) with or without LPS (1 ?g/mL).
Flow Cytometric Analysis
Leukocytes were first blocked with 10% v/v normal goat serum for
20 min at 4°C and then stained as described (17) with fluorescein-
conjugated mAb for 30 min at 4°C. Cells incubated with appropriate
isotype-matched immunoglobulin (BD PharMingen) served as nega-
tive controls. After staining, the cells were fixed in 2% v/v parafor-
maldehyde and then analyzed using an EPICS Elite flow cytometer
(Beckman Coulter, Hialeah, FL).
RNase Protection Assay
RNA was isolated from 5?106bead-purified kidney DC by an RNA
Isolation Kit (BD PharMingen). RNase protection assay (RPA) was
performed as described (17) using the RiboQuant Multi-Probe RPA
system (BD PharMingen). Kits containing cDNAs encoding mouse
IL-1R antagonist, IL-1?, IL-1?, interferon (IFN)-?, IFN-?, IL-6, IL-
10, IL-12p35, IL-12p40, migration inhibition factor, CC chemokine
receptor (CCR) 1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, and the
housekeeping genes L32 and glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) were used as templates for the T7 polymerase-
directed synthesis of phosphorus-32 uridine triphosphate-labeled an-
tisense RNA probes. Densitometric analysis was performed using a
GS-710 calibrated imaging densitometer (BioRad, Hercules, CA).
Individual gene expression was normalized to GAPDH expression by
division of the densitometric value for each gene by the correspond-
ing value for GAPDH and multiplying the product by 100.
Mixed Leukocyte Reaction
T cells from spleens of C3H mice were enriched (purity ?80%) by
passage through nylon wool columns (45 min; 37°C) and stimulated
with various numbers of ?-irradiated DC or bulk spleen cells as
In Vitro Induction and Functional Assessment of Alloreactive
The method used was modified after that described by Jonuleit et
al. (7). Freshly isolated (immature) or overnight LPS-matured, ?-ir-
radiated B10 renal DC were incubated with naive CD4?C3H splenic
T cells purified using CD4 immunomagnetic beads, as described by
the manufacturer (Miltenyi Biotech) at a ratio of 1:10 in 24-well
plates. Alloreactive T cells were expanded from day 6 in the presence
of recombinant murine IL-2 (50 U/mL; R&D Systems, Minneapolis,
MN). Two weeks after priming, T cells were restimulated with im-
mature or mature DC from the same donor strain under identical
conditions and by weekly repetitive stimulation thereafter. At vari-
ous times, three-color flow cytometric analysis was performed on
saponin-permeabilized cells (18) using anti-CD4 cytochrome c, anti-
CD25 FITC, and anti–IL-10 PE mAb (BD PharMingen) to determine
CD4?CD25?cells were immunobead-sorted using a CD4?CD25?
Treg cell isolation kit (Miltenyi Biotech) and added (5?105cells/well)
at the start of mixed leukocyte reaction (MLR) cultures, in which
various numbers of original donor (B10) or third-party (BALB/c)
stimulators were cultured with 2?105purified C3H responder T
balt5/ztr-tp/ztr-tp/ztr00704/ztr7010-04a marksjS?53/1/0414:194/Color Figure(s): 1,5Art: 145344
Vol. 77, No. 7
In Vivo Trafficking of DC
Immunobead-purified CD11c?DC (freshly isolated or LPS-ma-
tured) were labeled with the green fluorescent dye PKH-67 (Sigma)
according to the supplier’s instructions, then injected (2?106admin-
istered intravenously [IV]) into C3H mice through the lateral tail
vein. After 24 hr, the animals were killed and the spleens removed
for immunohistochemical analysis. Cryostat sections (8 ?m) were
stained with biotin-conjugated anti-CD3 (BD PharMingen) or anti-
MOMA1 mAb (Bachem Bioscience, Inc., King of Prussia, PA) fol-
lowed by Cy3-conjugated streptavidin (Jackson ImmunoResearch,
West Grove, PA) to identify T cells or macrophages, respectively, and
to ascertain the interrelationship between these cells and the traf-
ficking donor DC. Cell nuclei were stained with DAPI.
Ex Vivo Analysis of Host Anti-donor T-Cell Responsiveness
C3H mice were injected IV with 2?106immunobead-purified im-
mature or mature B10 kidney DC, 7 days before harvesting spleens
for assessment of anti-donor T-cell responsiveness. T cells (purity
?80%) were used as responders in ex vivo 2° MLR, or as effectors in
cytotoxic T lymphocyte (CTL) assays, as described (8).
Vascularized Cardiac Transplantation
Surgical procedures were performed under methoxyflurane (Pit-
man-Moore, Atlanta, GA) inhalational anesthesia. Intra-abdominal
vascularized heterotopic cardiac transplantation was performed as
described (8). Graft function was monitored by daily palpation. Total
cessation of cardiac contraction was defined as rejection that was
Graft survival between groups of animals was determined by
Kaplan-Meier analysis, and statistical significance was tested using
the log-rank statistic.
Detection and Characterization of Flt3L-Mobilized Renal
DC In Situ
Presumptive DC have been identified previously in normal
rodent (10, 19) and human kidneys (20) by immunohisto-
chemical staining for common leukocyte Ag (CD45) or MHC
class II. In keeping with these observations, staining of nor-
mal B10 mouse kidneys (Fig. 1) revealed rare, dendriform
CD11c?cells, in the medulla (between tubules) (Fig. 1F).
After 10 days of systemic administration of Flt3L, CD11c?
cells were much more numerous, both in the cortex and in the
medulla. Intraglomerular CD11c?cells were observed (Fig.
1A). Both single, isolated DC, and aggregates of CD11c?cells
were evident in the cortex and medulla (Fig. 1C and D).
These CD11c?cells were predominantly immature, display-
ing low levels of CD86 (Fig. 1A and B). Two-color immuno-
histochemical staining for CD11c and CD8? revealed both
CD11c?CD8??and CD11c?CD8??subsets, with the former
being more numerous in each renal compartment (Fig. 1C
and D). Staining for MHC class II and CD11c confirmed the
presence of double-positive cells in kidneys from Flt3L-
treated (Fig. 1E) and normal mice (Fig. 1F).
Isolation, Morphologic Analysis, and Further Phenotypic
Characterization of In Vivo-Mobilized Renal DC
DC were isolated from both normal and Flt3L-treated
mouse kidneys after enzymatic digestion of renal tissue (see
Materials and Methods). Using CD11c immunobeads, an av-
erage of 5.8?0.2?104DC/organ (n?35 mice) could be iso-
lated from the metrizamide-enriched nonparenchymal cell
population of normal B10 mouse kidneys. Using flow cyto-
metric analysis, three subsets were identified: classic
CD11c?CD8??myeloid DC, CD11c?CD8??B220?“lym-
phoid-related” DC, and CDllc?CD8??B220?presumptive
preplasmacytoid DC (21). The typical yield of CD11c?cells
from Flt3L-treated mouse kidneys was 1.1?0.3?166cells/
organ (n?40 mice), representing an approximately 20-fold
increase compared with normal kidneys. After immunobead
sorting, CD11c?cells were examined by light, transmission,
and scanning electron microscopy or immunostained and fur-
ther characterized by flow cytometric analysis. The imma-
ture, bulk CD11c?cells displayed typical DC morphologic
characteristics, with short cytoplasmic processes and promi-
nent intracytoplasmic vacuolar compartments (Fig. 1G). By
contrast, LPS-treated, overnight-matured renal DC devel-
oped prominent cytoplasmic processes/veils (Fig. 1H). Flow
cytometric analysis of the bulk CD11c?population from
Flt3L-treated mice confirmed three distinct populations of
CD8??CD8??and B220?cells as in normal mice (Fig. 2A
and C–E). The most common subset in the renal CD11c
population from Flt3L-treated mice was the CD8??popula-
tion (approximately 80%), with CD8??and B220?cells con-
stituting approximately 13% and 7%, respectively. Renal DC
from normal, untreated B10 mice were then compared to DC
isolated from Flt3L-treated animals, with respect to their cell
surface co-stimulatory molecule and MHC class II Ag (IAb)
expression. Freshly isolated bulk CD11c?kidney DC from
normal mice (Fig. 2B) and DC from Flt3L-treated animals
(Fig. 2C–E) each expressed moderate MHC class II and low
to moderate levels of co-stimulatory molecules, indicating
that DC mobilized into kidneys by Flt3L had not undergone
significant change compared with normal renal DC. Similar
levels of MHC II and co-stimulatory molecules were ex-
pressed by the three DC subsets from Flt3L-treated animals
(Fig. 2). After overnight culture in LPS, the CD8??and
CD8??DC markedly increased surface MHC II, CD40,
CD80, and CD86 (Fig. 2F and G).
Freshly Isolated Renal DC Increase Transcription of IL-
12p35 and p40 and CCR7 after LPS Stimulation
To further characterize the renal DC, the RPA was used to
examine the expression of genes encoding the Th1-promoting
cytokine IL-12 and other cytokines by both freshly isolated,
CD11c immunobead-purified, and overnight LPS-cultured
DC. As shown in Figure 3, freshly isolated bulk CD11c?DC
transcribed mRNA encoding IL-12p40 (but not IL-12p35),
IL-1?, IL-1Ra, IL-10, and migration inhibition factor. After
overnight culture with LPS, the renal DC produced message
for IL-12p35, an essential subunit of bioactive IL-12p70, and
expressed higher levels of IL-1?, IL-1Ra, IL-10, and IL-
12p40. Freshly isolated renal DC displayed mRNA tran-
scripts for the chemokine receptors CCR1, CCR2, and CCR5,
with low expression of CCR7. This chemokine receptor ex-
pression pattern has been reported typically in immature
DC. On overnight culture with LPS, the DC decreased mes-
sage for CCR1, CCR2, and CCR5, but increased CCR7 mRNA
(Fig. 3), a further indication of their maturation.
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COATES ET AL.
April 15, 2004
Freshly Isolated Renal DC Induce Only Weak Naive
Allogeneic T-Cell Proliferative Responses but Strongly
Stimulate T-Cell Proliferation after LPS Stimulation
Consistent with their immature phenotype and absence of
IL-12p35 gene expression, freshly isolated, Flt3L-mobilized
B10 (H2b) bulk CD11c?renal DC were poor stimulators of
naive allogeneic (C3H; H2k) T cells in MLR (Fig. 4A). These
mobilized DC induced levels of T-cell proliferation similar to
those induced by bulk normal resident renal DC (data not
shown). After overnight culture in LPS, they matured into
potent inducers of naive T-cell proliferation (Fig. 4A).
Freshly Isolated Renal DC Promote the Generation of
CD4?CD25?IL-10?T Cells In Vitro
Jonuleit et al. (7) reported the ability of in vitro-generated
immature human DC to induce CD4?Treg cells. To assess
whether they could induce cells with a similar phenotype,
freshly isolated, in vivo-mobilized, immature bulk B10 renal
DC were used to stimulate naive C3H CD4?T cells (see
Materials and Methods). Alloreactive T-cell lines were ex-
panded by addition of recombinant IL-2. As shown in Figure
4(B), immature kidney DC induced the expansion of a minor
CD4?CD25?IL-10?T-cell population that was not evident in
cultures of mature DC-stimulated T cells. Immunobead-
sorted CD4?CD25?cells from the former cultures sup-
pressed both donor (B10) and third-party (BALB/c) APC-
induced naive T-cell proliferation when added at the start of
MLR cultures (Fig. 4C).
Immature Renal DC Traffic In Vivo to Host Secondary
To investigate the in vivo migratory ability of freshly iso-
lated (immature) compared with mature B10 renal DC,
2?106PKH-67–labeled bulk CD11c?cells from kidneys of
Flt3-mobilized mice were injected IV into normal allogeneic
(C3H) recipients. Examination of cryostat sections of spleens
by fluorescence microscopy 24 hr later (Fig. 5) revealed that
the labeled renal DC had migrated predominantly to
MOMA1-staining marginal zones (immature DC) (Fig. 5A–C)
or CD3?T-cell areas (mature DC) (Fig.D and E) (periarte-
riolar sheaths) (Fig. 5A). The immunohistochemical staining
confirmed that the migratory DC (purified using CD11c im-
munobeads) did not co-localize with MOMA1?macrophages,
ruling out the possibility that their apparent trafficking was
attributable to ingestion by these cells. The superior ability of
mature renal DC compared with immature DC to migrate to
T-cell areas likely reflects the stronger expression of CCR7 on
the former cells (Fig. 3B).
Immature Renal DC Fail to Prime Allogeneic T Cells
To examine the capacity of immature kidney DC to prime
allogeneic T cells in vivo, freshly isolated or mature immu-
nobead-purified bulk CD11c?DC (B10) were injected IV into
naive allogeneic (C3H) mice. Seven days later, the animals
were killed and purified splenic T cells were restimulated in
vitro with irradiated donor bulk B10 splenocytes for 72 hr. In
contrast to T cells from animals infused with immature kid-
ney DC, which exhibited minimal ex vivo anti-donor reactiv-
ity, those from mice given mature kidney DC showed marked
proliferation (Fig. 5F). Similarly, ex vivo-stimulated T cells
cence staining in normal and Flt3L-treated B10 mouse kidneys.
Glomeruli are encompassed (broken circles). (A and B) Staining
for CD11c (pink) and CD86 (green) in (A) cortex and (B) medulla
of Flt3L-treated mice indicates that the DC are predominantly
immature. (Inset in A) Detail of a glomerulus; (inset in B) DC
between parallel adjacent tubules (demarcated by white lines),
the lumens of which are indicated by asterisks. (C and D) Dual-
labeling of CD11c?and CD8? (green) indicates that both dou-
ble-positive CD11c?CD8??(yellow) and CD11c?CD8??DC sub-
sets are present within the cortex (C) and medulla (D) of Flt3L-
treated animals. (insets in D) Higher power view of CD8??T
cells (green cytoplasm with blue nucleus) in association with
CD11c?DC (left) and CD11c?CD8??medullary DC (focal green
staining within the cytoplasm of CD11c?cells) (right). Single
CD8??cells are likely to represent isolated T cells. (E and F)
Coincident MHC class II (IAb) expression (green) on CD11c?DC
(yellow dual stain) was more evident in Flt3L-treated (E) com-
pared with normal kidney (F), in which CD11c?MHC class II?
cells were scarce (inset; a single double-positive cell is seen in
the interstitium between tubules, the lumens of which are in-
dicated by asterisks), consistent with previously published re-
ports (magnification ?200; insets ?600. (G and H) Giemsa stain
of freshly isolated, immature DC and overnight LPS-stimulated
DC. (G) Freshly isolated, bulk CD11c?DC show typical imma-
ture DC morphology, with short cytoplasmic processes and
prominent intracellular vacuoles; (H) overnight LPS-stimu-
lated DC (106cells/mL; 1 ?g/mL LPS) show extensive cytoplas-
mic projections (magnification ?1,000).
Identification of CD11c?cells by immunofluores-
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Vol. 77, No. 7
from mice injected with immature renal DC failed to exhibit
cytotoxic activity against donor-specific target cells. By con-
trast, potent CTL activity was exhibited by donor Ag-restim-
ulated T cells from mice primed in vivo with LPS-stimulated
mature renal DC (Fig. 5G).
Freshly Isolated Renal DC Prolong Organ Allograft
Immature donor BM- or spleen-derived DC infused sys-
temically before transplantation prolong allograft survival
(8, 9). To ascertain whether freshly isolated renal DC from
Flt3L-mobilized animals could similarly affect organ graft
survival, 2?106immunobead-sorted bulk CD11c?B10 DC
were injected IV into normal C3H recipients 7 days before
vascularized B10 heart transplantation. No immunosuppres-
sive treatment was administered. As shown in Figure 6,
mean graft survival time was prolonged significantly, from 9
days to 19 days (P?0.01). By contrast, graft survival times in
C3H mice that received donor mature renal DC, 7 days before
organ transplantation, were not affected significantly. To
determine the specificity of this effect, C3H recipients
treated with B10 immature renal DC 7 days before trans-
plantation were challenged with third-party (BALB/c) heart
grafts. Allograft survival was prolonged, indicating a nonspe-
cific effect, as has been reported previously for in vitro-cul-
tured bone marrow-derived DC in this strain combination (8).
As in normal kidneys, intrarenal Flt3L-mobilized DC were
distributed widely throughout the cortex and medulla and
exhibited phenotypes and ex vivo functions consistent with
immaturity. Three distinct populations of normal resident
and in vivo-mobilized DC were identified, confirming and
further characterizing the heterogeneous nature of murine
renal DC described previously (15). In addition to classic
(CD11c?CD8??) described in mouse lymphoid tissues (22),
we also identified, for the first time, intrarenal precursors of
murine plasmacytoid DC (CD11c?B220?CD11b?), an impor-
tant type-1 IFN-producing cell population that has been re-
ported to date only in lymphoid tissue (21).
At the gene transcription level, freshly isolated bulk kid-
ney DC also showed a cytokine and CCR expression profile
FIGURE 2. (A) DC subsets in normal B10 mouse renal tissue. (left
panel) CD11c?bulk renal DC isolated from the kidneys of eight
normal mice.(right panel)
CD11c?CD8??B220?DC subsets in the bulk renal DC population.
(B–G) Co-stimulatory molecule and MHC class II (IAb) expression
on (B) normal and (C–G) Flt3L-mobilized renal DC. (B) Freshly
isolated CD11c?DC from normal B10 kidneys display low to neg-
ligible CD40, moderate CD80 and CD86, and moderate to high IAb
expression; (C) Flt3L-mobilized CD11c?CD8??B220?DC; (D)
Flt3L-mobilized CD11c?CD8??B220?DC; and (E) Flt3L-mobilized
CD11c?CD8??B220?DC (all freshly isolated) show comparable
expression of co-stimulatory molecules and MHC class II. After
overnight culture in granulocyte-macrophage colony-stimulating
factor plus LPS (106cells/mL; 1 ?g/mL LPS), Flt3L-mobilized
CD11c?CD8??DC (F) and CD11c?CD8??DC (G) markedly in-
Isotype controls. Data are from one representative experiment of
at least three performed for each subset under each condition.
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COATES ET AL.
April 15, 2004
consistent with immaturity. Thus, these DC expressed
mRNA for IL-12p40 but not IL-12p35, the subunit required
for bioactive IL-12p70 (Fig. 3). When administered IV, allo-
geneic immature Flt3L-mobilized kidney DC migrated to sec-
ondary lymphoid tissue, a property thought to be critical for
the role of DC in Ag presentation and the elicitation of host
T-cell responses (1). In contrast, lymphoid tissue homing of
DC-bearing epithelium-derived self-Ag (apoptotic bodies) has
been linked to the maintenance of tolerance in the normal
steady state (4). Cross-presentation of Ag to DC resident in
secondary lymphoid tissue may play a significant role in this
process (2). Considered together, our data provide a more ex-
tensive characterization of renal DC than reported previously.
They confirm that, when freshly isolated, they are similar to
immature DC resident in topologically external sites (e.g., epi-
dermal Langerhans cells (23)), as well as to those in other solid
organs, in particular, the heart (15) and liver (24).
The present findings raise the issue of the role of specific
DC subsets versus the maturational status of renal DC in
relation to the functional capacity of these APC (influence on
alloreactive T cells and transplant survival). Because only
limited numbers of highly purified individual DC subsets
could be obtained, even from large numbers of kidneys, we
focused on the influence of the maturational status of bulk
DC on T-cell function. In this regard, it may be argued that a
more physiologically representative “intact” DC population
was examined. Our data suggest that the in vivo immuno-
regulatory activity of these cells is related to their immatu-
rity. We and others have shown that, like freshly isolated
immature DC, stably immature cultured DC generated in the
presence of various anti-inflammatory agents (transforming
growth factor-?, IL-10, dexamethasone, or nuclear factor ?B
anti-sense oligodeoxynucleotides) prolong graft survival (re-
viewed in Morelli and Thomson (3)). In the present study, the
capacity of renal DC to prolong allograft survival was lost
after their in vitro maturation. Thus, we believe that the
regulatory activity of the DC in vivo can be ascribed to their
immaturity. Our studies and those of others have shown that
specific splenic DC subsets (myeloid, “lymphoid-related,” or
preplasmacytoid) can prolong organ allograft survival, and
FIGURE 3. (A) Cytokine and (B) CCR mRNA expression in freshly isolated (left column) and overnight LPS-stimulated bulk
CD11c?renal DC (106cells/mL; 1 ?g/mL LPS) (right column) demonstrated by RNAse protection assay. (C and D) Correspond-
ing densitometric analyses of cytokine and CCR mRNA expression in freshly isolated (filled column) and overnight-cultured
DC (open column) are also shown. Results are arbitrary densitometric units relative to housekeeping gene (GAPDH) expres-
sion and are means?1 SD obtained from three separate experiments.
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Vol. 77, No. 7
that this is a function of immature cells (8, 9, 25, 26). The
ability of immature donor renal DC to prolong allograft sur-
vival may be a shared property of all three subsets.
Two sets of observations provide clues to the possible
mechanism(s) whereby immature DC prolong organ allograft
survival. First, with the exception of CD8??DC (9), freshly
isolated immature but not mature donor splenic DC achieve
this effect. Second, the tolerogenic effect appears to be criti-
cally dependent on the temporal relationship between DC
infusion and the transplant procedure. Thus, immature DC
given 7 days before transplantation prolong graft survival (8,
25), but this effect is lost when the cells are administered
earlier or later (25, 27). The current paradigm most likely to
explain this critical dependency is the induction of Treg cells.
Immature but not mature human allogeneic DC induce IL-
10–secreting CD4?T cells (7). Thus, repeated stimulation of
naive cord blood T cells with immature, monocyte-derived DC
generates IL-10–producing CD4?T cells with low prolifera-
tive capacity when restimulated with donor Ag. Importantly,
these CD4?cells elicited in response to immature DC inhibit
syngeneic T-cell proliferation after alloantigen challenge (7).
Similar effects may account for the present in vivo observa-
tions when immature kidney DC were administered to allo-
geneic recipients and their splenic T cells harvested 7 days
later. Restimulation of these T cells with donor Ag revealed
markedly reduced proliferative and CTL responses compared
with those of animals given mature kidney DC. In vitro
studies further demonstrated that, in the presence of exoge-
nous IL-2, freshly isolated renal DC could induce allogeneic
CD4?CD25?IL-10?T cells and that CD4?CD25?cells from
FIGURE 4. Freshly isolated (imma-
ture) renal DC are weak inducers
of allogeneic T-cell proliferation
and can induce CD4?CD25?IL-10?
cells. (A) allostimulatory capacity
for naive C3H T cells of ?-irradi-
(mature) B10 kidney
monds), compared with freshly
isolated (immature) kidney DC
(open squares). Data are from one
three performed. Proliferative re-
sponses to syngeneic (C3H) spleen
cells (filled squares) and alloge-
neic (B10) spleen cells (triangles)
are also shown. (B) CD4?C3H T
cells were cultured with immature
or mature bulk CD11c?B10 renal
DC (10:1), expanded with IL-2 (50
U), and restimulated as described
(see Materials and Methods). His-
tograms show intracellular stain-
ing for IL-10 in CD4?CD25?cells
only in alloreactive T-cell popula-
tions stimulated with immature
sorted CD4?CD25?T cells from
cultures stimulated with imma-
ture kidney DC as in (B), inhibited
B10 or BALB/c APC-induced syn-
geneic C3H T-cell proliferation
when added (5.0?103cells/well) at
the start of 72 hr of MLR cultures.
Results are from one experiment
representative of two performed.
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COATES ET AL.
April 15, 2004
these cultures could suppress syngeneic T-cell proliferative
responses in MLR, although in a nonspecific manner. This
evidence is consistent with the hypothesis that Treg cells
induced in vivo may play a role in the prolongation of allo-
graft survival in this model. In human studies, autologous
immature monocyte-derived DC induced Ag-specific, IL-10–
producing T cells when injected subcutaneously (28). These T
cells are of particular interest, as they inhibit the effector
function of Ag-specific CD8?T cells, an effect that could be
highly beneficial in transplantation.
The pronounced increase in functionally immature DC
with capacity to regulate alloimmunity within the kidneys of
hematopoietin-mobilized animals may have potential clinical
implications. The beneficial effect of simultaneous combined
heart and kidney transplantation on cardiac rejection in hu-
mans has been documented, and a role for renal-derived
donor DC in the reduced incidence of cardiac rejection has
been suggested (29). Thus, the use of growth factors such as
Flt3L to augment immature interstitial DC in donor kidneys
before living-donor renal transplantation may reduce the
incidence of acute rejection and dependency on immunosup-
pressive drugs. To achieve this goal, careful attention must
be paid to the maturation status of the donor DC before and
after transplantation, as exposure to maturation stimuli is
likely to enhance their allostimulatory capacity. In this con-
text, it is important to note the effects of conventional immu-
nosuppressive therapy on DC function. Thus, Hackstein et al.
(30) have demonstrated that rapamycin inhibits DC matura-
tion in vivo and confers tolerogenic properties on these APC,
in addition to its well-recognized T-cell suppressive effects.
Because administration of the co-stimulation blocking agent
anti-CD40L (CD154) mAb markedly potentiates immature
donor DC tolerogenicity (27), possible candidate combination
therapies that maximize DC tolerogenicity can be envisaged.
The novelty of the present findings lies in the direct com-
parative phenotypic and functional characterization of resi-
allogeneic spleens; immature DC
fail to prime recipients for anti-do-
nor T-cell responses. (A–E) Local-
ization of bulk CD11c?kidney DC
in spleens after their systemic in-
jection. Two million freshly iso-
lated or mature B10 (H2b) renal DC
were labeled with the green fluo-
rescent dye PKH-67 and injected
IV into C3H (H2k) recipients. After
24 hr, spleens were removed and
cryostat sections stained for CD3
(pan T cells; red) or the macro-
phage marker MOMA1 (metallo-
philic macrophages of the mar-
ginal zone; red). (A–C) Freshly
isolated renal DC localized within
the marginal zone, but did not ap-
pear to be taken up by MOMA1?
macrophages. (D and E) In con-
trast, overnight-matured renal DC
were identified within T-cell areas.
(F) Ex vivo restimulation of C3H
splenic T cells from mice injected
IV 7 days previously with imma-
ture or mature B10 bulk kidney DC
(2?106). (G) Similar differences in
served in CTL assays. Data are
from one experiment representa-
tive of at least five performed
midino-2-phenylinolole; A, B, and
D, magnification ?200; C and E,
Homing of renal DC to
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Vol. 77, No. 7
dent and mobilized DC in a parenchymal organ for which few
data exist regarding this important APC population. Addi-
tional novel features include the identification of lymphoid-
related and preplasmacytoid DC in the kidney and the ca-
pacity of freshly isolated renal DC to induce Treg cells in
vitro. These properties may account for the ability of imma-
ture allogeneic kidney DC, infused in the normal steady
state, to subvert subsequent anti-donor responses and to
prolong allograft survival.
Acknowledgments. The authors thank Alison J. Logar for skilled
technical assistance, the Immunex Corporation (now Amgen) for
providing Flt3L, Masanori Abe, M.D., for help with data analysis,
and Miriam Meade for proficient manuscript preparation.
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