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CD103− and CD103+ Bronchial Lymph Node
Dendritic Cells Are Specialized in Presenting
and Cross-Presenting Innocuous Antigen to
CD4 + and CD8+ T Cells
Maria-Luisa del Rio, Jose-Ignacio Rodriguez-Barbosa,
Elisabeth Kremmer and Reinhold Förster
J Immunol 2007; 178:6861-6866; ;
doi: 10.4049/jimmunol.178.11.6861
http://www.jimmunol.org/content/178/11/6861
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Copyright © 2007 by The American Association of
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References
The Journal of Immunology
CD103ⴚ and CD103ⴙ Bronchial Lymph Node Dendritic Cells
Are Specialized in Presenting and Cross-Presenting Innocuous
Antigen to CD4ⴙ and CD8ⴙ T Cells1
Maria-Luisa del Rio,* Jose-Ignacio Rodriguez-Barbosa,* Elisabeth Kremmer,†
and Reinhold Förster2*
T
he MHC class II molecules load peptides derived from
exogenous proteins that are acquired by endocytosis or
from internalized plasma membrane proteins. In contrast,
MHC class I molecules normally incorporate peptides derived
from proteins synthesized in the cytosol (reviewed in Refs. 1, 2).
Thirty years ago, Bevan (3) reported that minor histocompatibility
Ags could be transferred from cells to host APCs and, as a consequence, stimulate cytotoxic CD8⫹ T cell-mediated immune responses. This phenomenon was named “cross-priming” (3) and the
processing of exogenous Ag for MHC class I presentation has been
referred to as “cross-presentation” (reviewed in Ref. 2). For selfAgs, the CD8␣⫹ dendritic cell (DC)3 subset has been identified for
its capability to cross-present Ag to CD8⫹ T cells giving rise to
two possible different outcomes: cross-priming or cross-tolerization (3, 4). Cross-tolerance implicates the induction of CD8⫹ T
cell tolerance by cross-presentation of self-Ags; by contrast, crosspriming involves the activation of CD8⫹ T cells for the generation
of CTL effector cells. It has been shown recently that the capacity
of DC for cross-presenting Ags depends on their Ag-processing
properties, suggesting that the CD8␣⫹ DC subset has specialized
machinery for cross-presenting self-Ags that is probably absent or
*Institute of Immunology, Hannover Medical School, Hannover, Germany; and †Institute of Molecular Immunology, National Research Center, Munich, Germany
Received for publication January 9, 2007. Accepted for publication March 20, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Grant SFB587-B3 from the German Research Foundation (to R.F.).
2
Address correspondence and reprint requests to Dr. Reinhold Förster, Institute of
Immunology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover,
Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived
DC; LN, lymph node; brLN, bronchial LN; i.t., intratracheal(ly).
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
less effective in other DC subsets (5). In contrast to the situation
described for self-Ags, little is known about DC that cross-present
innocuous inhaled Ag for cross-tolerization.
A recent report has identified the CD103⫹ DC in mice as a
major DC population residing in the lung mucosa (6). CD103 (␣E)
is the ␣-chain of the ␣E␤7 integrin, which has been reported to be
essential for the adhesion of human and mice intestinal lymphocytes to epithelial cells through the interactions with E-cadherin
(7). CD103⫹ DC in the lung do not express the typical plasmacytoid
DC (B220, Gr-1) or lymphoid markers (CD8␣), but express high
amounts of CD11c, MHC class II, and low levels of CD11b (6).
We have previously demonstrated that CCR7 expression plays a
pivotal role for the mobilization of DC from the skin (8), the intestine (9), and the lung (10) to the respective draining lymph
nodes (LN). This migration is indispensable for the immunological
relevant transport of Ag to tolerize CD4⫹ T cells. Consequently,
CCR7-deficient mice fail to induce tolerance toward ingested (9)
or inhaled Ags (10).
Based on these observations, we hypothesized that the impaired
migration of lung-derived DC may also influence cross-presentation of innocuous Ag and the subsequent Ag-driven CD8⫹ T cell
proliferation. In agreement with this concept, we now demonstrate
that the adoptive transfer of OVA-loaded CCR7-deficient bone
marrow-derived DC (BMDC) to C57BL/6 (B6) recipients, which
also received CFSE-labeled OT-I cells, failed to induce proliferation of the adoptively transferred OT-I cells. Of interest, OT-I
proliferation could be rescued when B6 BMDC were transferred
intratracheally (i.t.) to CCR7-deficient recipients. Further analysis
of this process revealed that Ag-carrying CD11chighCD11blow
CD8␣⫺CD103⫹ DC, present in the brLN of wild-type and plt/plt,
but almost absent in CCR7-deficient mice, have evolved to acquire
specialized functions for cross-priming innocuous Ag to CD8⫹ T
cells under tolerogenic conditions. In contrast, CD11cintCD11bhigh
CD103⫺ DC are specialized in presenting innocuous Ag to CD4⫹
T cells.
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Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8ⴙ T lymphocytes through MHC class I,
a process referred to as cross-presentation. In this study, we demonstrate that CD103ⴙ (CD11chighCD11blow) and CD103ⴚ
(CD11cintCD11bhigh) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions
in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103ⴚ DC are specialized in presenting innocuous Ag
to CD4ⴙ T cells, whereas CD103ⴙ DC, which do not express CD8␣, are specialized in presenting Ag exclusively to CD8ⴙ T cells.
In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103ⴙ DC are largely absent in the brLN, although CD103ⴙ DC are
present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8ⴙ T cells can be activated under
tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103ⴙ brLN DC are specialized in
cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7ⴚ/ⴚ mice, our findings strongly
suggest that brLN CD103ⴙ DC are lung-derived and that expression of CCR7 is required for their migration from the lung into
its draining lymph node. The Journal of Immunology, 2007, 178: 6861– 6866.
CD103⫹ DC AND CROSS-PRESENTATION
6862
Materials and Methods
Mice
CCR7-deficient mice (11) were backcrossed for eight generations to
C57BL/6 mice. C57BL/6 Ly5.2 (CD45.2), plt/plt, OT-I Thy1.1, and OT-II
Ly5.1 mice, which carry a transgenic TCR for the H2-Kb-restricted SIINFEKL
peptide derived from OVA257–264 or for the H2-Ab-restricted ISQAVHAA
HAEINEAGR peptide derived from OVA323–339, were also used in this study.
All the mice strains were bred and maintained under specific pathogen-free
conditions at the Central Animal Facility of Hannover Medical School
(Hannover, Germany) or were purchased from Charles River Breeding
Laboratories. All animal experiments were conducted in accordance
with local and institutional guidelines.
Generation of OVA-loaded BMDC
BMDC of wild-type and CCR7-deficient mice were generated as previously
described (8). Briefly, bone marrow cells were harvested from femurs and
tibiae and subsequently cultured in complete medium supplemented with 30
ng/ml GM-CSF. At day 3, culture medium was removed and the cells were
selected by adherence. Four days later, cells were incubated for 24 h with 100
␮g/ml OVA grade VI (Sigma-Aldrich) and 1 ␮g/ml LPS (Sigma-Aldrich). At
day 8, BMDC were washed extensively with PBS and i.t. injected.
Mice were anesthetized i.p. with 0.2 mg of ketamine and 0.02 mg of xylazine per gram of body weight. A blunt cannula (0.7 ⫻ 19 mm, Introcan;
B. Braun) was gently introduced through the larynx into the trachea under
visual control (Head-worn Loupe KS; Zeiss). A volume of 60 ␮l of extensively washed OVA-loaded BMDC or Cy5-labeled OVA (1 mg/ml in
PBS) was i.t. injected. All batches of OVA were tested for the presence of
LPS (LAL QCL 1000; BioWhittaker-Cambrex), and only batches containing ⬍5 EU/mg were used.
Isolation of brLN DC
The thoracic cavity was dissected and the brLNs were exposed, isolated,
and minced. DC were released from the brLNs by enzymatic digestion
using RPMI 1640 containing 25 ␮g/ml DNase I (Roche), 10% FCS, 25
mM HEPES, and 500 ␮g/ml collagenase D (Roche) for 30 min at 37°C on
an orbital shaker at 200 rpm. The cell suspension obtained was filtered
using a nylon mesh. Finally, the cells were washed twice with PBS containing 2% FCS and 2 mM EDTA.
FIGURE 1. Phenotypical characterization of Ag-bearing CD103⫹ and
CD103⫺ DC of the brLN. A, Cy5-labeled OVA was i.t. injected to wildtype mice, and 24 h later the brLN were dissected and digested with
collagenase D-DNase I. The cell suspension was labeled with mAbs
against CD11c, CD3, CD19, and CD103. CD11c⫹CD3⫺OVA-Cy5⫹
CD19⫺ DAPI-negative were sorted into CD103⫹ and CD103⫺ DC. B,
Uptake of soluble Cy5-labeled OVA Ag by CD103⫹ and CD103⫺ DC
in the brLN. Cells were gated on CD11c⫹CD103⫹ propidium iodidenegative (PI⫺) and CD11c⫹CD103⫺ propidium iodide-negative stain
and analyzed for the expression of OVA-Cy5.
Flow cytometry
The DC of brLNs were prepared following the protocol described. All
the cell suspensions were preincubated with 2 ␮g/ml blocking anti-FcR
(2.4G2) before staining to reduce nonspecific binding. The following
mAbs were used: CD3 (17A2), CD4 (RMCD4-2), CD8␤ (RMCD8-2),
CD8␣ (53-6.7), CD11b (M1/70.15), CD11c (HL3), CD205 (NLDC-145),
and CD19 (1D3) were purified and labeled in our laboratory; CD2 (RM2-5),
CD18 (C71/16), CD11a (I21/7), CD80 (16-10A1), CD86 (RMMP-1), CD49d
(428), CD49e (5H10-27), PDL1 (MIH3), PDL2 (TY25), CD40 (3/23), rat
isotype control (R35-95), MHC class II (IAb) (AF6-120.1), Ly6c (AL-21),
V␣2 (B20.1), V␤5 (MR9-4), and CD103 (M290) were purchased from BD
Biosciences; 33D1 was obtained from eBioscience. Before i.t. transfer, BMDC
differentiation was monitored applying mAbs to CD11c, IAb, and CD86. The
brLN cell suspensions were stained with anti CD3, CD19, CD11c, and CD103
mAbs. Consequently, CD11c⫹CD103⫹OVA-Cy5⫹CD3⫺CD19⫺ as well as
CD11c⫹CD103⫺OVA-Cy5⫹CD3⫺CD19⫺ DC were sorted on a FACSAria
cell sorter (BD Biosciences). The purity of sorted DC was always ⬎96%.
Dead cells and debris were excluded by propidium iodide or DAPI (4⬘,6⬘diamido-2-phenylindole hydrochloride) staining. Flow cytometry acquisition
was conducted on a LSR-II cytometer (BD Biosciences) while data analysis
was performed using WinList 5.0 (Verity Software House).
Aerosol treatment
In some experiments, mice were treated in an aerosol chamber with OVA
aerosol (1% in water) or water alone. At 24 h after of adoptive transfer of
CFSE-labeled cells, OVA solution or water was vaporized for 20 min using
a PariBoy vaporizer as previously described (10). On day 4, mice were
sacrificed and the brLN as well a nondraining LN were analyzed by flow
cytometry to monitor the rate of T cell division.
In vitro T cell proliferation
Mice i.v. received 60 ␮g of OVA-Cy5 in PBS in a total volume of 60 ␮l.
The brLN were removed 24 h later and CD3⫺CD19⫺CD11c⫹OVACy5⫹CD103⫹ DC or CD3⫺CD19⫺CD11c⫹OVA-Cy5⫹CD103⫺ DC were
isolated by flow sorting. DC (2000 cells) were seeded in U-bottom 96-well
plate and cocultured with 15 ⫻ 104 OT-I or OT-II T cells. Proliferation of
OT-I and OT-II T cells in triplicate wells was measured after 3 days by
pulsing the cells with 1 ␮Ci/well [methyl-3H]thymidine followed by further
incubation for 16 h. Plates were harvested, and thymidine uptake was quantified in a beta counter (Microbeta TriLux; PerkinElmer).
Results
Adoptive transfer of OVA-specific T cells from TCR
transgenic mice
Phenotypical characterization of Ag-loaded CD103⫹ and
CD103⫺ DC in the lung-draining LN
Spleen and peripheral LN of OT-I Thy1.1 or OT-II Ly5.1 mice were
minced through a nylon mesh. Cells were washed twice with PBS and
adjusted according to the number of V␣2⫹V␤5⫹ cells. A total of 5 ⫻
107cells/ml were labeled with 5 ␮M CFSE (Molecular Probes) in PBS at
37°C for 10 min. The reaction was stopped by adding 2 volumes of cold
RPMI 1640 containing 10% FCS followed by two washes in PBS. Each
recipient received i.v. 15 ⫻ 106 OT-I T cells into the tail vein.
CD103 is expressed on 30 –50% of the lung-derived CD11c⫹ DC
migrating to the brLN (10). Thus, similar to the situation described
for DC of the mesenteric LN (12) and lung (6), CD103⫹ and
CD103⫺ DC are present in the brLN of wild-type mice following
i.t. instillation of 60 ␮g of Cy5-labeled OVA (Fig. 1A). Of interest,
CD11c was expressed differentially on these DC, being more
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Intratracheal instillation of OVA and Ag-pulsed DC in vitro
The Journal of Immunology
6863
FIGURE 2. CD103⫹ and CD103⫺ DC in the brLN do not express markers
characteristic for plasmacytoid DC or CD8␣ DC subsets. A, Cy5-labeled OVA
was i.t. administered to wild-type mice and 24 h later cells were isolated from
the brLN. Cell suspensions were prepared from collagenase D-DNase I digested brLN. DC from brLN were gated on CD11c⫹OVA-Cy5⫹CD103⫹ propidium iodide-negative (PI⫺) or CD11c⫹OVA-Cy5⫹CD103⫺ propidium iodide-negative stain and analyzed for the expression of DC markers or
costimulatory and adhesion molecules as indicated. CD103⫹ DC (gray shaded
histogram) and CD103⫺ DC (open histogram) are shown. B, Expression of
CD8␣ and 33D1 on CD11c⫹ from brLN (top panels) and spleen (bottom
panels). Isotype control is shown by dotted line histogram. C, CD11c⫹OVACy5⫹CD103⫹ or CD11c⫹OVA-Cy5⫹CD103⫺ DC from brLN were analyzed
for the expression of CD205 (top panels). Spleen cells from untreated wildtype mice were gated on CD11c⫹CD103⫹ or CD11c⫹CD103⫺ and analyzed
for the expression of CD205 (bottom panels). Isotype control is shown by
dotted line histogram.
abundantly present in the CD103⫹ DC (Fig. 1A). Assessing the
capacity for Ag uptake and presentation, CD103⫹ and CD103⫺
DC were analyzed for the presence of OVA-Cy5⫹. OVA-Cy5 was
found to be trapped with roughly equal efficiency by both CD103⫹
and CD103⫺ DC (Fig. 1B).
CD11c⫹OVA-Cy5⫹CD103⫹ and CD11c⫹OVA-Cy5⫹CD103⫺
DC of the brLN were analyzed for the expression of surface markers. Interestingly, CD103⫹ DC expressed a low amount of the
myeloid marker CD11b, whereas this marker was considerably
brighter in CD103⫺ DC. A reverse situation could be observed for
marker CD11a (LFA-1), which was more abundantly expressed on
CD103⫹ DC than in CD103⫺ DC. Further analysis of expression
levels of costimulatory (CD40, CD80, CD86, PD-L1, PD-L2,
MHC class II) and adhesion molecules (CD18, CD49d, CD49e,
CD2, CD54, CD102, CD106) failed to reveal any significant differences between the two subsets (Fig. 2A). CD103⫹ and CD103⫺
DC were also negative for B220, CD4, Ly6c (Fig. 2A and data not
shown) and CD8␣ (Fig. 2B), indicating that neither CD103⫹ nor
CD103⫺ DC belong to the CD8␣⫹ DC or plasmacytoid DC subset. CD11c⫹ DC isolated from spleen were used as positive controls because they contain subpopulations positive for CD8␣ or the
33D1 Ag (Fig. 2B). Of interest, DC isolated from the brLN were
negative for CD8␣ as well as 33D1 (Fig. 2B), but CD103⫹ and
CD103⫺ DC expressed CD205 to some degree (Fig. 2C).
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FIGURE 3. CD103⫺ and CD103⫹ brLN DC are specialized in presenting and cross-presenting innocuous Ag. CD11c⫹OVA-Cy5⫹CD103⫹ (f)
and CD11c⫹OVA-Cy5⫹CD103⫺ (䡺) DC were flow-sorted from the brLN
of wild-type animals following 24 h after i.t. instillation of Cy5-labeled
OVA. CD103⫹ or CD103⫺ DC (2000/well) were cocultured with 15 ⫻ 104
OT-I (A) or OT-II (B) T cells in triplicate as indicated. Three days later,
cultures were pulsed with 1 ␮Ci/well [3H]thymidine for 16 h before thymidine uptake was quantified. Data are mean ⫾ SD for n ⫽ 3 independent
experiments.
6864
CD103⫹ DC AND CROSS-PRESENTATION
CD103 expression distinguishes two DC subsets specialized in
presenting or cross-presenting Ag to T cells under tolerogenic
conditions
Besides the presentation of pathogen-derived Ags, airway DC are also
capable of presenting inhaled innocuous Ags to T cells to induce
tolerance. We therefore aimed to determine whether different airway
DC subsets have evolved to become specialized in presenting harmless Ag to distinct T cell subsets. To that end, OVA-Cy5 was i.t.
instilled in wild-type mice. After 24 h, CD11c⫹OVA-Cy5⫹CD103⫹
and CD11c⫹OVA-Cy5⫹CD103⫺ DC were isolated by flow sorting
from the brLN and cocultured in vitro with OT-I or OT-II T cells for
72 h before T cell proliferation was assessed by [3H]thymidine incorporation. Strikingly, CD103⫹ and CD103⫺ DC displayed distinct capabilities in priming CD4⫹ or CD8⫹ T cells. Thus, OVA-Cy5⫹
CD103⫹ DC efficiently cross-presented the OVA Ag to CD8⫹ T
cells, whereas OVA-Cy5⫹CD103⫺ DC almost lacked such stimulatory activity (Fig. 3A). Conversely, CD103⫺ DC induced massive
proliferation of CD4⫹ OT-II, but not CD8⫹ OT-I T cells (Fig. 3B).
Although both CD103⫹ and CD103⫺ DC exhibited similar competency in taking up innocuous Ag (Fig. 1B), these DC process and
present Ag differently to CD4⫹ and CD8⫹ T cells.
earlier (10), this observation suggests that the migration of
CD103⫹ DC from the lung to the brLN depends on CCR7 expression on lung-residing DC. Because plt/plt mice still express the
CCL21-leu gene in lymphatic endothelial cells of the lung, it
seems likely that CCL21-Leu allows for the migration of CD103⫹
DC from the lung to its draining LN in this mouse strain.
We next sorted CD11c⫹OVA-Cy5⫹CD103⫹ DC and CD11c⫹
OVA-Cy5⫹CD103⫺ DC from the brLN of wild-type and plt/plt
mice 24 h after i.t. instillation of Cy5-labeled OVA. As in
In vivo OVA-loaded brLN CD103⫹ DC from wild-type and
plt/plt mice exhibit similar efficiency in cross-presenting
innocuous Ag to CD8⫹ T cells
Because OVA-Cy5⫹CD103⫹ DC in wild-type animals are specialized in cross-presenting innocuous Ag, we were interested in
assessing the presence of this cell population in the brLN of
CCR7-deficient and plt/plt mice. The latter mouse strain represents
a naturally occurring mutant that lacks expression of CCR7 ligands
in LN but not in nonlymphoid organs such as the lung. We determined the percentage of CD11c⫹OVA-Cy5⫹CD103⫹ DC in collagenase-digested brLN of wild-type, CCR7-deficient, as well as
plt/plt mice 24 h after i.t. instillation of OVA-Cy5. As depicted in
Fig. 4A, we failed to observe any significant differences between
wild-type and plt/plt mice regarding the frequency of OVA-loaded
CD103⫹ DC. In contrast, the frequency of this cell population was
severely reduced in CCR7⫺/⫺ mice. Together with data published
FIGURE 5. CCR7-deficient mice are deficient, whereas plt/plt mice are
proficient in cross-presenting inhaled Ag. CFSE-labeled OT-I cells were
adoptively transferred into wild-type, plt/plt, or CCR7-deficient recipients.
Subsequently, mice received an OVA or a control (water) aerosol. After 4
days, the proliferation of V␣2⫹CD8⫹ brLN T cells of mice that received
the OVA (left) and water (right) was analyzed. Data are representative of
two experiments with two to three mice each per experiment.
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FIGURE 4. OVA-Cy5-loaded CD11c⫹CD103⫹ DC cross-present the OVA Ag to the same extent in plt/plt mice as in wild-type mice. A, The percentage
of CD11c⫹OVA-Cy5⫹CD103⫹ DC of all cells present in brLN of wild-type (n ⫽ 11 animals) (䡺), plt/plt (n ⫽ 6 animals) (f), and CCR7-deficient (n ⫽
5 animals) (s) mice was analyzed 24 h after i.t. instillation of OVA-Cy5 (mean ⫾ SD). n.s., Not significant. ⴱ, p ⬍ 0.05. CD11c⫹OVA-Cy5⫹CD103⫹
and CD11c⫹OVA-Cy5⫹CD103⫺ DC were flow-sorted from the brLN of wild-type and plt/plt mice 24 h after i.t. instillation of OVA-Cy5. As indicated,
CD103⫹ or CD103⫺ DC (2000 per well) were cocultured in triplicates with 15 ⫻ 104 OT-I (B) or OT-II (C) T cells for 3 days before they were pulsed
with [3H]thymidine for 16 h. Proliferation was analyzed applying a beta counter. Data are mean ⫾ SD with results from one experiment. Similar results
were obtained in a second experiment.
The Journal of Immunology
6865
with the previously reported data (10), these results strongly suggest that the lack of CCR7 prevents the migration of DC from the
lung to the draining brLN and, because of that, CCR7-deficient
mice fail to present and cross-present Ag within the brLN to OT-II
and OT-I T cells, respectively.
To test this hypothesis, mature OVA-loaded BMDC from wildtype or CCR7-deficient mice were i.t. instilled into wild-type or
CCR7-deficient recipients that adoptively received CFSE-labeled
OT-I T cells 1 day earlier. As expected, OVA-loaded wild-type DC
induced a strong proliferation of the OT-I T cells in the draining brLN
of wild-type (Fig. 6A, top panels) and CCR7-deficient (Fig. 6B, top
panels) mice. In contrast, OVA-loaded CCR7-deficient DC i.t. instilled in wild-type (Fig. 6A, bottom panels) or CCR7-deficient (Fig.
6B, bottom panels) mice failed to induce significant proliferation of
OT-I T cells. Under neither condition was the proliferation of cells in
the nondraining inguinal LN observed (Fig. 6).
Discussion
wild-type animals, only CD103⫹ but not CD103⫺ DC from plt/plt
mice efficiently cross-presented the OVA Ag to CD8⫹ T cells in
vitro (Fig. 4B). Likewise, CD11c⫹OVA-Cy5⫹CD103⫺ DC from
plt/plt preferentially stimulated the proliferation of CD4⫹ T cells
(Fig. 4C). In contrast to plt/plt and wild-type mice, CCR7-deficient
mice displayed a pronounced impaired migration of CD103⫹ DC
from the lung to the brLN and consequently they were largely
absent in this lymphoid compartment (10). For this reason, the
described experiment could not be performed using brLN DC of
CCR7-deficient animals. However, as outlined in the following
sections, the role of brLN DC in T cell proliferation could be
addressed in CCR7-deficient mice in vivo.
CCR7⫺/⫺ mice are deficient whereas plt/plt mice are proficient
in cross-presenting innocuous inhaled Ag in vivo
We subsequently compared the in vivo capacity of DC to crosspresent inhaled Ag. To that end, wild-type, plt/plt, and CCR7deficient mice were i.v. injected with CFSE-labeled OT-I T cells
and 1 day later received an OVA aerosol. After an additional 4
days, the lung-draining LN was removed and analyzed. In wildtype and plt/plt mice, OVA-aerosol treatment induced Ag-specific
proliferation of OT-I T cells, whereas Ag-induced proliferation
was not detectable at all in CCR7-deficient mice (Fig. 5). Together
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FIGURE 6. The i.t. instilled CCR7-deficient OVA-loaded BMDC fail
to induce CD8⫹ T cell proliferation in the brLN under tolerogenic conditions. As indicated, BMDC from wild-type or CCR7-deficient mice were
loaded with OVA protein during maturation and i.t. instilled into wild-type
(A) or CCR7-deficient (B) mice that had adoptively received CFSE-labeled
OT-I cells 24 h earlier. Three days after DC instillation, the proliferation of
the adoptively transferred V␣2⫹CD8⫹ T cells was analyzed in the lungdraining brLN or the nondraining inguinal LN. Representative results are
shown for two to seven mice per group.
The rapid mobilization of maturing DC from the site of Ag encounter to the draining lymphoid tissue is a critical event for the
rapid initiation of adaptive immune responses (13–16). Chemokines and chemokine receptors have been identified as essential
players regulating DC mobilization and migration (8, 11, 17, 18).
In particular, upon maturation, DC up-regulate the expression of
CCR7 and become responsive to CCL19 and CCL21 (19). Consequently, CCR7 expression is essential for the mobilization of DC
from the skin (8), the intestine (9, 12), and the lung (10) to the
respective draining LN to induce tolerance. As a result of this
impaired DC migration, CCR7-deficient mice fail to induce tolerance toward ingested (9) or inhaled Ags (10). In this study, we
demonstrate that under tolerogenic conditions, expression of
CD103 distinguishes two DC subsets that differentially prime
CD4⫹ or CD8⫹ T cells in the lung-draining LN.
The lung of wild-type and CCR7-deficient mice contain
CD103⫹ DC that are reduced in the brLN of CCR7-deficient mice
compared with wild-type mice (6, 10). CCR7-deficient mice also
show a reduced number of these DC in skin-draining LN (data not
shown) and in the mesenteric LN (12), suggesting that, in general,
immature DC residing between epithelia might require CCR7 expression for proper mobilization from the periphery to the draining
LN. In contrast to CCR7-deficient mice, the percentage of Agcarrying CD103⫹ DC is not significantly reduced in the brLN of
plt/plt mice. Although this mutant lacks expression of CCR7 ligands in lymphoid organs, one isoform of CCL21 (CCL21-Leu) is
still present in these mice in nonlymphoid organs such as the lung,
particularly in the endothelium of the afferent lymphatics (20).
Therefore, it seems conceivable that expression of CCL21-Leu on
afferent lymphatic vessels in the lung of plt/plt mice is sufficient to
allow for steady state migration of DC to the brLN in these mice.
Based on the differential expression of CD103, this study demonstrates the existence of two distinct DC subsets that have
evolved different capabilities in priming CD8⫹ or CD4⫹ T cells in
the brLN under tolerogenic conditions. In addition to CD103, these
two DC subsets also express different levels of ␣-integrins such as
CD11a, CD11b, and CD11c. However, these subsets were indistinguishable regarding the expression of costimulatory molecules
or any of the other markers tested in this study. Despite the fact
that CD103⫹ and CD103⫺ DC express similar amounts of costimulatory molecules, their interaction with T cells leads to
largely opposing outcomes. CD103⫹ DC primarily cross-present
Ag to CD8⫹ T cells, whereas CD103⫺ DC prime CD4⫹ T cells in
both wild-type and plt/plt mice, but not in CCR7-deficient animals.
The distinct capabilities of the CD103 DC subsets to prime T cells
seems not to be related to their ability to take up Ag because both
6866
Acknowledgments
We are grateful in particular to Günter Bernhardt and Oliver Pabst for
valuable suggestions on the manuscript, Matthias Ballmaier and Christina
Reiner for cell sorting, and Jasmin Bölter for expert technical assistance.
Disclosures
The authors have no financial conflict of interest.
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DC subsets capture comparable amounts of OVA following i.t.
instillation. Along this line, it has been recently shown that spleenderived CD8␣⫹ and CD8␣⫺ DC engulfed similar amounts of Ag,
yet within the spleen, only the CD8␣⫹ DC subset is able to crosspresent Ag to CD8⫹ T cells, whereas the CD8␣⫺ DC more efficiently stimulates CD4⫹ T cell proliferation (5, 21). Furthermore,
during the submission process of this report Dudziak et al. (22)
reported that, within the spleen, only CD205⫹ DC are able to
cross-present Ag, whereas another DC population, negative for
CD8␣ but positive for the 33D1 Ag, is specialized for presentation
on MHC class II. Of interest, this study could show that the ability to present or cross-present Ag is intrinsic to the DC subsets and
this role is associated with increased expression of components of
the MHC class II and MHC class I processing pathways, respectively. Remarkably, the CD8␣⫹ DC subset is largely absent in
nonlymphoid tissues, such as skin (23), pancreas (24), stomach
(25), and lung (26), and its contribution to the pool of migrating
DC from the lung to the brLN seems to be of negligible relevance
(10, 26, 27). Data provided in this study support this idea because
CD103⫹ DC that cross-present Ag in the brLN lack CD8␣ expression. Along this line, the present study reveals that none of
markers successfully used to discriminate presenting (33D1⫹)
from cross-presenting (CD8␣⫹, CD205⫹) DC in the spleen can be
used in the lung-draining brLN. Steady-state DC isolated from this
lymphoid organ lack expression of CD8␣ as well as 33D1, and both
the CD103⫹ as well as the CD103⫺ DC weakly express CD205.
It is noteworthy to mention that several reports have recently
suggested that CD103⫹ and CD103⫺ DC isolated from the lung or
the intestine might exhibit similar competence in stimulating
OVA-specific CD4⫹ and CD8⫹ T cells (6, 12, 28). However, those
studies were performed pulsing CD103⫹ and CD103⫺ DC in vitro
with OVA peptides. Applying this procedure, peptides are directly
loaded to the MHC molecules from outside, circumventing the
need for Ag uptake, processing, and presentation. In contrast, in
the present study DC were loaded in vivo by i.t. application of the
entire OVA protein. Together, these data suggest that both
CD103⫹ and CD103⫺ DC can present Ag to CD4⫹ as well as
CD8⫹ T cells as long as the appropriate peptides gain access to the
MHC groove. Because CD103⫹ and CD103⫺ brLN DC serve opposing functions with regard to presentation and cross-presentation
of exogenous Ag once loaded in vivo, it seems likely that both cell
types may exhibit profound differences regarding Ag processing as
recently described for CD205⫹ and 33D1⫹ DC of the spleen (22).
In summary, our data reveal that Ag-carrying CD103⫹ brLN DC
are specialized in cross-presenting innocuous inhaled Ag. Because
these cells are largely absent in CCR7-deficient but not plt/plt
mice, our data strongly suggest that brLN CD103⫹ DC are lung
derived and that expression of CCR7 is required for their migration
from the lung into its draining LN.
CD103⫹ DC AND CROSS-PRESENTATION