The Journal of Experimental Medicine
JEM © The Rockefeller University Press $30.00
Vol. 204, No. 13, December 24, 2007 3133-3146 www.jem.org/cgi/doi/10.1084/jem.20071733
C-type lectin receptors are predominantly ex-
pressed by DCs and function as pattern recog-
nition receptors by interacting with glycosylated
moieties on pathogens. This usually results in
internalization of the pathogen, lysosomal deg-
radation, and subsequent loading of pathogen-
derived peptides into MHC molecules for
antigen presentation ( 1 ). Langerin (CD207) is a
type II lectin receptor containing only one car-
bohydrate recognition domain ( 2, 3 ). Although
the ligands of langerin have not been clearly
identifi ed, langerin has recently been shown to
bind HIV-1 gp120 through calcium-dependent
mannose recognition ( 4 ). Langerin is also a po-
tent inducer of Birbeck granules, a hallmark of
epidermal DCs, also called Langerhans cells
(LCs), which consist of zippered membranes
that are visible by electron microscopy ( 2 ). Upon
antigen capture, langerin associates to Birbeck
granules and facilitates the routing of captured
mannosylated ligands from the cell surface into
a nonclassical antigen-processing pathway ( 2 ).
Therefore, expression of the langerin receptor
is likely to refl ect a functionally distinct cell
population with specifi c antigen uptake and
Langerin is expressed at high levels on
mouse and human LCs ( 2, 3 ), but is absent on
dermal interstitial DCs ( 5 ), providing a unique
tool to study the biology of cutaneous DC
populations. Cutaneous DCs include langerin +
MHC class II + LCs in the epidermis ( 6 ) and
langerin – MHC class II + interstitial DCs in the
dermis ( 7 – 10 ). In addition to interstitial DCs,
the dermis contains migratory langerin + MHC
class II + LCs en route to the skin draining LNs
(DLNs) ( 11 – 14 ). In the LNs, DCs are catego-
rized as blood-derived CD8 + and CD8 – DCs
and migratory DCs recruited from the periphery
Abbreviations used: EGFP,
enhanced GFP; DLN, draining
LN; DT, diphtheria toxin; DTR,
DT receptor; HEV, high endo-
thelial venule; LC, Langerhans cell.
A. Kissenpfennig ’ s present address is Infection and Immunity
Group, Center for Cancer Research and Cell Biology, School
of Biomedical Sciences, Queen ’ s University, Belfast,
The online version of this article contains supplemental material.
Blood-derived dermal langerin + dendritic
cells survey the skin in the steady state
Florent Ginhoux , 1,2 Matthew P. Collin , 1,2 Milena Bogunovic , 1,2
Michal Abel , 1,2 Marylene Leboeuf , 1,2 Julie Helft , 1 Jordi Ochando , 3
Adrien Kissenpfennig , 4 Bernard Malissen , 4 Marcos Grisotto , 1
Hans Snoeck , 1 Gwendalyn Randolph , 1 and Miriam Merad 1,2
1 Department of Gene and Cell Medicine and 2 Department of Medicine, Mount Sinai School of Medicine,
Mount Sinai School of Medicine, New York, NY 10029
3 Unidad de Imunologia de Transplantes, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220 Madrid, Spain
4 Centre d ’ Immunologie de Marseille-Luminy, Institut National de la Sant é et de la Recherche M é dicale U631,
Centre National de la Recherche Scientifi que UMR6102, Universit é de la M é diterrann é e, 13288 Marseille Cedex 9, France
Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens.
Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as
migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse
model that allows conditional ablation of langerin + cells in vivo, together with congenic
bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived
dendritic cells (DCs), we have revisited the origin of langerin + DCs in the skin DLNs. Our
results show that in contrast to the current view, langerin + CD8 – DCs in the skin DLNs do
not derive exclusively from migratory LCs, but also include blood-borne langerin + DCs
that transit through the dermis before reaching the DLN. The recruitment of circulating
langerin + DCs to the skin is dependent on endothelial selectins and CCR2, whereas their
recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show
that circulating langerin + DCs patrol the dermis in the steady state and migrate to the skin
DLNs charged with skin antigens. We propose that this is an important and previously
unappreciated element of immunosurveillance that needs to be taken into account in the
design of novel vaccine strategies.
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
Figure 1. Kinetics of langerin + repopulation in the epidermis and in skin DLNs. 6 – 8-wk-old Langerin-DTR/EGFP mice received a single intraperito-
neal injection of DT (1 ? g) and the kinetics of reappearance of langerin + was followed by fl ow cytometry in the epidermis and the skin DLNs at different
time points after depletion (Fig. S1). (A, top) Dot plots show I-A b and langerin expression (EGFP) among living (DAPI ? ) CD45 + cells in the epidermis at
different times after DT treatment. The remaining CD45 + I-A b ? langerin-EGFP ? cells represent dendritic epidermal T cells (DETC). (A, bottom) The percentage
of cells expressing CD8 and langerin among DAPI ? CD11c + I-A b+ DCs is shown for skin DLNs. (B) Graphs in log scale present the kinetics of reappearance of
JEM VOL. 204, December 24, 2007
the skin and remain of host origin > 18 mo after initiation of
the parabiosis ( 24 ). Therefore, expression of the congenic allele
in each parabiont and in recipients of congenic BM grafts can
be used to trace the blood- versus LC-derived origin of skin
DLN langerin + DCs. We have excluded from our analysis
LN langerin lo CD8 + DCs, as they are thought to derive from
blood circulating precursors. In contrast, skin DLN langerin hi
CD8 – DCs are thought to derive exclusively from epider-
mal LCs and were the focus of this study. Our results reveal for
the fi rst time the presence in mice of a population of langerin +
DCs, independent of LCs, in the dermis and the skin DLNs.
Our study also reveals that LC-independent langerin + DCs
are recruited from the blood to the dermis in the steady state
to capture tissue antigens before emigrating to the skin DLNs,
where they present skin-derived peptide-MHC complexes.
Repopulation of langerin + DCs in skin DLNs precedes
the repopulation of LCs in the epidermis
Injection of DT into Langerin-DTR/EGFP mice leads to
elimination of langerin + DCs in the absence of overt skin or
systemic toxicity, and therefore can be used to examine the
repopulation of langerin + DCs. As previously described, DT
eliminated the entire LC pool in the epidermis, in addition to
langerin lo CD8 + DCs and langerin hi CD8 – DCs in the skin
DLNs ( Fig. 1, A and B ). LCs were totally eliminated 2 d after
DT injection and remained absent > 14 d after treatment in
the epidermis ( Fig. 1, A and B ). Langerin + DCs in the skin
DLNs were also effi ciently depleted after DT, but in contrast
to LCs, their repopulation occurred much earlier ( Fig. 1,
A and B ). Langerin + DCs in the skin DLNs include CD8 + DCs
and CD8 – DCs. Langerin lo CD8 + DCs recovered to normal
levels in < 5 d after DT administration. Their rapid repopula-
tion after DT administration is consistent with the fact that
langerin lo CD8 + DCs are thought to derive from circulating
precursors with a turnover time of a few days ( 25, 26 ). In
contrast, langerin + CD8 – DCs, a population that is thought to
derive exclusively from epidermal LCs, started to repopulate
the skin DLNs long before LCs reappeared in the epidermis
( Fig. 1, A and B ), accounting for 25 – 30% of total langerin + CD8 –
DCs in normal untreated skin DLNs. Langerin + CD8 – DCs
were not detected in nonskin DLNs, such as mesenteric LNs,
after DT treatment (unpublished data). Based on these results,
we conclude that a population of LC-independent langerin +
DCs, distinct from CD8 + DCs, exists in the skin DLNs. The
level of langerin expression on repopulating DCs in the skin
DLNs was measured by EGFP fl uorescence and by langerin
through the lymphatics ( 15 ). In the skin DLNs, migratory
DCs include langerin + LCs and dermal langerin – DCs ( 14, 16 ).
However, it is now clear that in mice, langerin expression is
not restricted to LCs, but is also expressed on CD8 + DCs.
Indeed, CD8 + langerin lo DCs are present in all lymphoid or-
gans, including LN, spleen, and thymus ( 3, 17, 18 ). In con-
trast to migratory LCs, CD8 + DCs express much lower levels
of langerin and lack Birbeck granules, suggesting a non-LC
origin of these cells ( 19 ). Therefore, in skin DLNs, langerin +
cells are thought to include CD8 + langerin lo blood-derived
DCs and CD8 – langerin hi LC-derived DCs. Langerin expres-
sion has also been recently identifi ed on DCs in the lung mucosa
and vascular wall ( 20 ) and in the gut serosa ( 21 ).
Although the mechanisms that regulate the traffi cking of
tissue DCs to the DLN during infl amed conditions have been
well studied ( 22 ), much less is known about the traffi cking of
DC precursors from the blood to tissue and from tissue to
DLN in the steady state. In this study, we have revisited the
origin and homeostasis of langerin + DCs in the skin DLNs in
the absence of overt tissue injury. To examine the homeosta-
sis of langerin + DCs, we used a recently described mouse
model that allows the specifi c depletion of langerin + cells,
while sparing other DC populations. This model consists of
a knock-in mouse expressing enhanced GFP (EGFP) fused
with a diphtheria toxin (DT) receptor (DTR), under the con-
trol of the langerin promoter (Langerin-DTR/EGFP) on the
C57BL/6 background ( 18, 23 ). Administration of DT leads
in 24 h to the elimination of all langerin + cells, including
LCs, without aff ecting the langerin – DC compartment and
without skin or systemic toxicity ( 18, 23 ). After toxin treatment,
LCs repopulate the epidermis in 6 – 8 wk, off ering a window
of time to investigate the relative contribution of DC precur-
sors to the skin DLN DC compartments ( 18, 23 ). The original
description of this model showed that the repopulation of
langerin + DCs in the skin DLNs precedes LC repopulation in
the epidermis by weeks ( 18, 23 ). Intrigued by these results, we
sought to further clarify the origin and the homeostasis of
skin DLN langerin + DCs.
We have previously used congenic parabiotic pairs and
congenic BM chimeric mice as tools to diff erentiate epider-
mal LCs from circulating DC precursors. We have established
that in mice that have been lethally irradiated and reconsti-
tuted with donor congenic BM, epidermal LCs remained of
host origin throughout life, despite a full donor engraftment
in the blood ( 24 ). We also demonstrated that in congenic
parabiotic mice that share the same blood circulation but
separate organs for long period of time, LCs do not mix in
LCs in the epidermis and CD8 ? langerin + ( ? ) versus CD8 + langerin lo ( ? ) in the skin DLNs. Mean values ( ± the SD) are shown. (right) Histogram shows the
percentage of CD8 ? langerin + (mean values ± the SD) among CD11c + I-A b+ cells before and after DT treatment (DAY14). (C) Overlaid histograms show the
langerin levels of expression measured either by EGFP fl uorescence (top) or langerin intracellular staining (bottom) in skin DLN CD11c + I-A b+ CD8 ? langerin +
cells before and after DT treatment (DAY14). C57BL/6 control and isotype control are shown in black for Langerin-EGFP or langerin intracellular staining,
respectively. Representative data from three independent experiments are shown. Each experiment included at least three separately analyzed mice per
time point; error bars represent the SD between the results obtained from each of the three mice. (D) Double immunostaining (B220 in blue and langerin
in red) of skin DLNs from Langerin-DTR/EGFP (Langerin-EGFP in green) before and after DT treatment (DAY14). Fig. S1 is available at http://www.jem.org/
cgi/content/full/jem.20071733/DC1. Bars: (top) 200 ? m; (bottom) 50 ? m.
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
intracellular staining. LC-independent langerin + DCs in the
skin DLNs expressed higher levels of langerin compared with
CD8 + DCs and similar levels compared with skin DLN lan-
gerin + DCs in untreated mice ( Fig. 1 C ). Using immuno-
staining and confocal analysis of DT-treated skin DLNs, we
were able to visualize in situ the presence of LC-independent
langerin + DCs in the T cell areas of the skin DLNs 2 wk after
DT, a time when no LCs are present in the skin ( Fig. 1 D ).
The repopulation of langerin + CD8 – DCs in skin DLNs
correlates with the recruitment of circulating langerin + DCs
to the dermis
Langerin + CD8 – DCs in the skin DLNs can derive either from
the skin or from blood circulating precursors that enter
direc tly into the LN from the blood. Because LCs are absent
when langerin + CD8 – DCs reappear in the skin DLNs, we looked
for langerin + DCs in the dermis. We detected langerin + DCs
in the dermis long before LCs reappeared in the skin ( Fig. 2,
A and B ). Dermal langerin + DCs appeared around day 5 and
increased up to day 10 after DT injection, after which they
remained stable in the dermis, reaching 20% of total dermal
langerin + cells, 10% of total dermal DCs, and 2% of total der-
Dual origin of langerin + CD8 – DCs in parabiotic animals
The aforementioned results suggest that a population of
langerin + DCs, independent of LCs, resides in the dermis and
in the skin DLNs. To directly examine the origin of skin DLN
langerin + DCs, we used parabiotic mice in which C57BL/6
CD45.2 + mice were paired with congenic CD45.1 + mice so
that they shared the same blood circulation, but separate or-
gans, for prolonged periods of time. As previously described,
epidermal LCs remained of recipient origin > 6 mo after para-
biosis, which is consistent with their origin from skin-resident
precursors ( Fig. 3, A and B ). Therefore, the presence of
CD45.1 + langerin + DCs in the dermis of the CD45.2 + parabi-
ont should establish their independence from LCs and their
recruitment from the blood to the dermis in the steady state.
As shown in Fig. 3 , we were able to detect blood-derived
langerin + CD8 – DCs in the skin ( Fig. 3, A and B ) and the skin
DLNs ( Fig. 3, C and D ) of each parabiont. Total dermal DC
chimerism was low, but signifi cant, compared with LC chi-
merism as previously described ( 27 ), refl ecting either prolonged
or partial self-replenishment ( 27 ) or, as recently suggested, an
uneven distribution of DC precursors ( 26 ). Nevertheless, the
presence of donor-derived dermal langerin + DCs suggests
that, in contrast to LC-derived dermal DCs, dermal langerin +
DCs can originate from blood-derived precursors in the steady
state. Interestingly, the chimerism of langerin + DCs was lower
than langerin ? DCs. Because host langerin + DCs consist of a
mixture of dermal langerin + DCs and migrating LCs, it is
likely that the lower chimerism of langerin + compared with
langerin – DCs refl ects the contribution of migrating LCs.
Therefore, to better reveal the recruitment of blood-derived
langerin + cells to the skin and to the skin DLNs, we parabi-
osed Langerin-DTR/EGFP CD45.2 + C57BL/6 animals
with CD45.1 + C57BL/6 and followed the repopulation of
langerin + DCs in the CD45.2 + parabionts at diff erent times
after DT treatment. 2 d after DT injection, langerin + DCs
were eliminated from the skin and the skin DLNs of the
CD45.2 + Langerin-DTR/EGFP parabionts (unpublished data).
18 d after DT administration, a time point where LCs are absent
( Fig. 3 E , top), the percentage of donor CD45.1 + langerin +
DCs increased to the level of langerin – DCs in the dermis
Figure 2. Kinetics of langerin + repopulation in the dermis. Kinetics of reappearance of langerin + DCs was followed by fl ow cytometry in the dermis.
(A) Dots plots show langerin expression (EGFP) among live (DAPI ? ) CD45 + I-A b+ cells in the dermis at different times after DT treatment. (B) Graph in log
scale presents the percentage of I-A b+ langerin + DCs among total dermal DAPI ? CD45 + cells ( ? ) versus epidermal LCs ( ? ) at different times after DT treat-
ment. (right) Histogram shows the percentage of I-A b+ langerin + DCs among DAPI ? CD45 + cells before and after DT treatment (DAY14). Mean values ( ± the SD)
are shown. Representative data from three independent experiments are shown. Each experiment included at least three separately analyzed mice per
time point; error bars represent the SD between the results obtained from each of the three mice.
JEM VOL. 204, December 24, 2007
scribed, a few weeks after transplant, most blood leukocytes
originate from donor BM – derived precursors and express
CD45.1, whereas the majority of epidermal LCs are main-
tained by radioresistant precursors, and remain of host
(CD45.2 + ) origin throughout life, in the absence of skin in-
jury ( 24 ) ( Fig. 4 B ). Using fl ow cytometry analysis of skin
samples isolated at diff erent times after transplant, we found
that a substantial population of langerin + DCs in the dermis of
BM chimeras expressed donor CD45.1 antigens, suggesting
that they derived from donor BM-derived circulating precur-
sors and not from the host remaining CD45.2 + LCs ( Fig. 4,
A and B ). Consistent with our results ( Fig. 2 ), this population
corresponded to ? 10% of dermal MHC class II + cells. Blood-
derived CD45.1 + langerin + DCs in the dermis were also MHC
class II + , CD11c int , CD11b + , CX 3 CR1 – , CD8 – , a phenotype
similar to that expressed by classical LCs with the exception
( Fig. 3, E and F , bottom) and in the skin DLNs ( Fig. 3, G and H )
of the CD45.2 + Langerin-DTR/EGFP parabionts. These re-
sults establish that in the steady state, circulating langerin + DCs
are recruited to the dermis and to the skin DLNs indepen-
dently of LCs.
The origin of langerin + CD8 – DCs in congenic BM chimeras
Congenic BM chimeric mice provide another model in
which the CD45 congenic allele can be used to distinguish
LCs from blood-derived DCs in the skin DLNs. The advan-
tage of this model is that the majority of circulating leuko-
cytes express the congenic allele, whereas in parabiotic mice,
no more than 50% of leukocytes express the congenic allele
( 26 ). To generate congenic BM chimeras, C57BL/6 CD45.2 +
mice were lethally irradiated and reconstituted with BM cells
isolated from C57BL/6 CD45.1 + mice. As previously de-
Figure 3. Homeostasis of langerin + DCs in parabiotic mice. Each parabiotic pair consisted of one CD45.1 + C57BL/6 mouse with either a CD45.2 +
C57BL/6 mouse (A – D) or CD45.2 + Langerin-DTR/EGFP C57BL/6 mouse for DT depletion experiments (E – H). (A – D) Skin and skin DLN chimerism was exam-
ined by fl ow cytometry in untreated CD45.2 + parabiont of a CD45.1/CD45.2 parabiotic pair at 2 mo after the initiation of parabiosis. (A) Dot plots show
the chimerism of gated LCs (CD45 + I-A b+ langerin + ) in the epidermis (top) and the chimerism of gated langerin – and langerin + DCs (CD45 + I-A b+ ) in the
dermis (bottom). (B) Bar graphs show the percentages of donor (CD45.1 + )-derived cells among gated LCs (top) and langerin – or langerin + dermal DCs
(bottom). (C) Dot plots show the percentage of host and donor cells among gated CD11c + I-A b+ DCs, langerin – CD8 – DCs and langerin + CD8 – DCs. (D) Mean
values of langerin – versus langerin + DCs chimerism in CD45.2 + parabionts. Similar data are found in the skin and skin DLNs of untreated CD45.2 Langerin-
DTR/EGFP parabionts (unpublished data). Representative data from 5 separate experiments are shown. Each experiment included two to four mice. Skin
was separately analyzed for each mouse, whereas skin DLNs were pooled (pool of two mice); error bars represent the SD between the results obtained
from each mouse (skin) or from pooled DLNs (skin DLN). (E – H) Similar data to that found in A – D was obtained from DT-treated CD45.2 + Langerin-DTR/
EGFP parabiont of a CD45.1/CD45.2 Langerin-DTR/EGFP parabiotic pair. Data presented are from 18 d after DT treatment, a time when LCs are still de-
pleted (top), whereas langerin + dermal DCs are recovered. Representative data from two separate experiments are shown. Each experiment included two
separately analyzed mice, except for the skin DLNs that were pooled. For the skin, error bars represent the SD between the results obtained from each of
the two mice.
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
Figure 4. Homeostasis of langerin + DCs after congenic BM transplantation. Lethally irradiated host CD45.2 + C57BL/6 were transplanted with con-
genic donor CD45.1 + C57BL/6 BM (CD45.1 into CD45.2 chimeric mice). 2 mo after reconstitution, host versus donor chimerism was measured in the
langerin + DC population in the skin (A and B) and in skin DLNs (D and E). (A) Dot plots show I-A b and langerin expression among dermal CD45 + cells, and the
host versus donor chimerism of langerin + dermal DCs (CD45 + I-A b+ ). (B) Mean values of langerin + DCs chimerism in the dermis. LC (CD45 + I-A b+ langerin + )
chimerism in the epidermis is shown as control. (C) Expression of different markers was compared by fl ow cytometry between host remaining (CD45.2 +
I-A b+ langerin + ) LCs and donor-derived langerin ? or langerin + dermal DCs. (D) Dot plots show CD8 versus langerin expression among CD11c + I-A b+ cells in
the skin DLNs and host versus donor chimerism of CD8 ? langerin + DCs. (E) Mean values of langerin + DC chimerism. (F) Phenotype of donor-derived CD8 ?
DC langerin + , CD8 + DC langerin ? , and host remaining LCs. (G) Back skin cross-sections isolated from CD45.2 Langerin-EGFP into CD45.1 chimeric mice
(left) or the reverse CD45.1 into CD45.2 Langerin-EGFP chimeric mice (right) were stained with anti-langerin antibody. Nuclei were counterstained with
DAPI. Bar, 50 ? m. (H) Dermal cell suspensions were prepared from CD45.2 Langerin-EGFP into CD45.1 chimeric mice. Donor langerin + dermal DCs were
sorted on the criteria of CD45.2, I-A b , and EGFP expression and harvested onto cytospin slides for langerin staining. Inset shows langerin staining control.
Bar, 10 ? m. Representative data from four independent experiments are shown. Each experiment included at least three separately analyzed mice; error bars
represent the SD between the results obtained from each of the three mice.
JEM VOL. 204, December 24, 2007
langerin + CD8 – DCs. These results suggest that circulating
langerin + CD8 – DCs reach the skin DLNs through the skin and
not directly from the blood.
Mechanisms that regulate the recruitment of circulating
langerin + DCs to the dermis
Previous results suggest that circulating DC precursors roll
continuously along noninfl amed mouse dermal endothelium
in vivo, an interaction shown to be strictly dependent on
endothelial selectins ( 31 ). To examine if endothelial selectins
play a role in the homeostasis of dermal langerin + DCs, we re-
constituted lethally irradiated C57BL/6 mice that lack both
P- and E-selectins (P/E-selectin KO), or wild-type C57BL/6
controls with BM cells isolated from congenic CD45.1 + C57BL/6
mice. 2 mo after reconstitution, we found that P/E-selectin KO
have similar numbers of CD45.1 + circulating B cells, mono-
cytes, and neutrophils compared with wild-type controls, sug-
gesting that absence of P- or E-selectins did not aff ect BM
engraftment (unpublished data). In contrast, the percentage of
dermal langerin + DCs in P/E-selectin KO mice was 30 – 50%
lower than the percentage of dermal langerin + DCs in wild-
type mice ( Fig. 5 E ). Similar results were observed for dermal
langerin – DCs (unpublished data).
We previously established that CCR2 chemokine ligands
are required for the recruitment of circulating dermal DC
precursors to the skin ( 27 ). We also established that CCR2
and CCR6 chemokine ligands are required for the recruit-
ment of circulating LC precursors and the repopulation of
LCs in infl amed skin ( 32, 33 ). To examine the role of these
molecules in the recruitment of circulating donor langerin +
DC precursors to the dermis, we reconstituted lethally irradi-
ated CD45.1 + C57BL/6 mice with a 1:1 mixture of hemato-
poietic progenitors cells that consisted of CD45.2 + BM cells
that either lacked CCR2 (CCR2 KO) or CCR6 (CCR6 KO)
and wild-type CD45.1 + BM. 2 mo after reconstitution, we
found that CCR2 ? / ? , CCR6 ? / ? , and wild-type CD45.1 +
BM gave rise to similar numbers of circulating B cells and
neutrophils, suggesting that the absence of CCR2 and CCR6
did not aff ect BM engraftment (unpublished data). In contrast,
CCR2 ? / ? CD45.2 + CD115 + circulating monocytes repre-
sented 30 – 40% of circulating CD45.2 + CCR6 ? / ? or CD45.1 +
wild-type monocytes (unpublished data), confi rming that ab-
sence of CCR2 expression on hematopoietic progenitors af-
fects monocyte repopulation in the blood ( 27, 34 ). We found
that CCR2 ? / ? dermal langerin + DCs were reduced in the
skin compared with their wild-type counterpart ( Fig. 5 F ),
whereas CCR6 KO langerin + dermal DCs were not aff ected
( Fig. 5 F ). Similar results were observed for dermal langerin –
DCs ( Fig. 5 F ). These diff erences underline the essential role
of CCR2 in the recruitment of dermal langerin + DCs to the
skin, whereas the role of CCR6 seems to be restricted to the
repopulation of epidermal LCs.
Role of langerin + CD8 – DCs in skin immunity
Our results reveal for the fi rst time that circulating langerin +
DC precursors are recruited to the dermis in the steady state
of the langerin levels, but diff erent from that of dermal lan-
gerin – DCs ( Fig. 4 C ). We also discovered that, in the skin
DLNs, an increased proportion of langerin + CD8 – DCs ex-
press CD45.1 (60 – 70%), and therefore are derived from
blood-precursors and not from epidermal LCs ( Fig. 4, D and E ).
Blood- and epidermal-derived langerin + CD8 – DCs were
indistinguishable phenotypically and expressed higher levels
of MHC class II and langerin and lower levels of CD11c
compared with langerin lo CD8 + DCs ( Fig. 4 F ). Interestingly,
langerin expression formed a smear on dermal blood – derived
langerin + DCs, which may suggest that the langerin receptor
was being acquired upon entry in the dermis and up-regu-
lated during the migration to the skin DLNs. These results are
consistent with the data from a companion paper showing
that blood-derived langerin + DCs that emigrate from skin ex-
plants have similar langerin levels compared with LCs ( 28 ).
Donor-derived langerin + DCs were visualized in situ by
confocal microscopy analysis of skin sections isolated from
(CD45.2 + Langerin-EGFP BM→CD45.1 + C57BL/6) or the
reverse (CD45.1+C57BL/6 BM→CD45.2+Langerin-EGFP)
mice chimeras ( Fig. 4 G ). Donor-derived langerin + DCs ap-
pear scattered in the dermis, without any conspicuous local-
ization near the hair follicle. Donor-derived CD45.2 + MHC
class II + Langerin-EGFP + DCs were also purifi ed by FACS
and stained for langerin on cytospins ( Fig. 4 H ). Altogether, our
results suggest that, although undistinguishable phenotypi-
cally, blood- and epidermal-derived langerin + CD8 – DCs coexist
in the skin DLNs of BM chimeric mice.
Mechanisms that regulate the traffi cking of langerin + CD8 –
DCs to the skin DLNs
The presence of blood-derived langerin + CD8 – DCs in the skin
DLNs always paralleled their presence in the skin. However,
the percentage of blood (CD45.1 + )-derived langerin + DCs
among total langerin hi DCs was always higher in the skin DLNs
compared with the dermis (compare Fig. 4 B with Fig. 4 E ),
suggesting that these cells either transit more rapidly than mi-
gratory LCs, but do not accumulate in the skin, or that they
can also be recruited to the skin DLNs directly from the blood.
To address these questions, we reconstituted lethally irradiated
CD45.1 + C57BL/6 recipient mice with BM cells isolated from
congenic CD45.2 + mice that lack L-selectin (CD62L KO) or
the chemokine receptor CCR7 (CCR7 KO). L-selectin is
constitutively expressed on most leukocytes and plays an im-
portant role in leukocyte homing into lymphoid tissue from
the blood ( 29 ). CCR7 is a chemokine receptor that plays a
critical role in the migration of DCs from the skin to the DLNs
( 30 ); therefore, absence of CCR7 should interfere with the
migration of DCs from the skin to the skin DLNs. 2 mo after
reconstitution, the percentage of CCR7 KO and CD62L KO
circulating langerin + DCs in the dermis was similar to their
wild-type counterpart ( Fig. 5, A and B ). In contrast, the per-
centage of CCR7 KO langerin + CD8 – DCs was markedly re-
duced compared with CCR7 +/+ langerin + CD8 – DCs in the
skin DLNs ( Fig. 5, C and D ). No diff erences were observed
between the percentages of CD62LKO versus CD62L +/+
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
Figure 5. Mechanisms involved in the recruitment to the dermis and the migration to the skin DLNs of dermal langerin + DCs. (A – D) Host
congenic CD45.1 + C57BL/6 were lethally irradiated and transplanted with donor CD45.2 + C57BL/6 (Control), CCR7-defi cient (CCR7 KO), or CD62L-defi cient
(CD62L KO) BM. After complete donor engraftment, the chimerism of total langerin + cells in the dermis and in the skin DLNs was measured by fl ow
cytometry. (A) Dot plots show CD45.1 (host) versus CD45.2 (donor) expression in langerin + dermal DCs from control, CCR7KO, and CD62LKO chimeric mice.
(B) Mean values ( ± the SD) of langerin + dermal DCs chimerism. (C and D) Same as in A and B, but for CD8 ? langerin + DCs in the skin DLNs of control, CCR7
KO, or CD62L KO groups. (E) Lethally irradiated host wild-type CD45.2 + C57BL/6 (Control) or CD45.2 + P/E-selectin defi cient (P/E-selectin KO) were lethally
irradiated and transplanted with congenic CD45.1 + C57BL/6 BM. After complete donor engraftment, the chimerism of total CD45 + I-A b+ langerin + DCs in
the dermis was measured by fl ow cytometry. Bar graph shows the chimerism of langerin + dermal DCs in control group (black bars) versus P/E-selectin
KO group (white bars) 2 mo after transplantation. Mean values ( ± the SD) are shown. (F) CD45.1 + C57BL/6 were lethally irradiated and reconstituted with
a mixture of CD45.1 + WT BM and CD45.2 + BM isolated from WT, CCR2 KO, or CCR6 KO BM, as described in the Materials and methods. 2 mo after trans-
plant, chimerism of total langerin + DCs in the dermis was measured. Bar graph shows the mean value of the percentage of CD45.2 + cells among langerin +
and langerin ? dermal DCs in control group (black bars), CCR2 KO group (white bars), and CCR6 KO group (gray bars). Mean values ( ± the SD) are shown.
Representative data from two independent experiments are shown. Each experiment included at least three separately analyzed mice; error bars represent
the SD between the results obtained from each of the three mice.
or after minor injuries (i.e., DT or radiation injuries). Our
results also suggest that circulating langerin + precursors that
are recruited to the dermis do not establish in the epidermis,
but emigrate to the skin DLNs. To examine if blood-derived
dermal langerin + DCs play a role in skin immunity, we sought
to analyze whether they can capture, process, and present
skin-derived antigen in the skin DLNs under steady-state
conditions. To assess antigen presentation, we took advan-
tage of the YAe monoclonal antibody that recognizes an MHC
class II I-E – derived peptide presented in the context of MHC
class II I-A b molecules. We reconstituted lethally irradiated
F1 (C57BL/6 I-A b – / – x BALB/c I-E d+ ) recipient mice with
BM cells isolated from CD45.1 + C57BL/6 I-A b+/+ Langerin-
EGFP BM. In transplanted mice, only I-A b+/+ donor cells,
and not host residual LC I-A b ? / ? , can present I-E d – de-
rived peptide recognized by YAe. In the skin of F1 (C57BL/
6xBALB/c) animals, I-E d is expressed by host radioresistant
LCs ( 24 ) and some activated keratinocytes ( 35 ) (unpub-
lished data). Therefore, donor I-A b+/+ DCs should be able to
present I-E d – derived peptides and stain positive for YAe only
if they can capture, process, and present skin-derived anti-
gens. We found that, in the dermis, donor-derived circulating
JEM VOL. 204, December 24, 2007
such as the neck and the back skin. However, the constant
percentage of donor-derived langerin + in skin DLNs draining
diff erent parts of the skin, including those containing host LCs
exclusively, argues against this hypothesis (unpublished data).
Alternatively, the radiation-induced injuries that might occur
in transplanted BM chimera mice may also aff ect the homeo-
stasis of dermal langerin + DCs and increase their recruitment
to the skin DLNs. Another possibility is that blood-derived
langerin + DCs are recruited directly from the blood to the skin
DLNs. Several lines of evidence argue against this possibility.
First, the presence of langerin + CD8 – DCs in the skin DLNs
always paralleled their presence in the skin, with the latter re-
constituting the dermis before the skin DLNs in depletion ex-
periments. Second, their recruitment to the skin DLNs was
independent of the L-selectin (CD62L), but required the che-
mokine receptor CCR7. CD62L is constitutively expressed on
most leukocytes, including DC precursors, and plays a critical
role in the fi rst steps of leukocyte extravasation from the blood
to the LN, allowing the rolling of blood leukocytes on high
endothelial venules (HEVs) ( 29 ). CCR7 is the receptor for
two structurally related chemokines, CCL19 and CCL21 ( 37 ).
CCL19 and CCL21 are expressed by stromal cells within the
lymphoid T cell zones, and CCL21 is expressed by HEVs and
at lower levels by the lymphatic endothelium ( 38, 39 ). In mice
lacking CCR7 ( 30 ) or CCR7 ligands ( 40 ), DCs fail to migrate
from the skin to the T cell areas of the skin DLNs, providing
the most compelling evidence for an essential role of CCR7 in
this process. Altogether, these studies led to the current con-
cept that migrating DCs express CCR7 and become chemo-
tactically attracted to CCL21-expressing lymphatic vessels. The
migrating DCs reach the subcapsular sinus of the DLNs and
follow a gradient of CCL21 and CCL19 to move into the
lymphoid T zone ( 38 ). Unlike all other chemokine receptors,
CCR7 is resistant to ligand-induced down-regulation ( 41 ),
which may explain how DCs can use CCR7 to perform all
the critical steps of migration. The failure of blood-derived
langerin + DCs to enter the skin DLNs in the absence of CCR7,
but not in the absence of CD62L, strongly suggests that these
cells reach the skin DLNs through the skin lymphatics and not
directly from the HEVs. However, CD8 + DCs, a population
thought to be recruited to the LN through the HEVs, were
also able to reach the skin DLNs in the absence of CD62L;
however, in contrast to CD8 – langerin + DCs, the lack of
CCR7 did not aff ect their recruitment to the skin DLNs (un-
published data). These results were surprising, as we expected
that, similar to naive T cells ( 39 ), expression of CD62L and
CCR7 would both be required for effi cient homing to the
HEVs. However, these data support previous fi ndings, show-
ing that CD62L is not required for the migration of spleen
DCs on resting or activated endothelial cells in vitro ( 42 ), and
suggest that other molecules might orchestrate the recruitment
of blood DCs through the HEVs. A potential candidate might
be “ chemerin, ” a proteolytically regulated chemoattractant
protein expressed at high levels by HEVs and shown to che-
moattract in vitro ChemR23 + DCs, which include human
blood DCs, but not LCs ( 43, 44 ).
dermal langerin + DCs stained positive for YAe, as did langerin –
dermal DCs, establishing their capacity to capture and pres-
ent exogenous skin-derived antigens ( Fig. 6, A and B , top).
Our results also show that blood-derived dermal langerin +
DCs that migrate to the skin DLNs also stain positive for YAe
( Fig. 6, A and B , bottom). A higher percentage of blood-
derived dermal langerin + DCs were positive for YAe in the
skin DLNs compared with the dermis ( Fig. 6, A and B , bottom),
suggesting that dermal-derived langerin + DCs process and
present skin-derived antigens en route to the skin DLNs. Similar
results were obtained when corresponding purifi ed dermal
DC populations were examined on cytospins for langerin and
YAe expression ( Fig. 6 C ).
Using three separate models to trace the turnover of circulat-
ing leukocytes, our study reveals the presence of a population
of langerin + DCs, independent of LCs, in the dermis and skin
DLNs of mice in the steady state.
We used parabiotic mice to establish the constitutive re-
cruitment of blood-derived langerin + DCs to the dermis and
their subsequent emigration to the skin DLNs. Parabiotic
mice provide a useful model to study the physiological turn-
over of leukocytes ( 36 ). Although recent data suggest that
circulating DC precursors do not reach complete equilibrium
in parabionts ( 26 ), this model continues to be very useful to
compare traffi cking patterns among DC subsets. Parabiotic
mice are particularly useful to distinguish LC-derived DCs
from blood-derived dermal langerin + DCs in the steady state
because LCs fail to mix in the epidermis > 18 mo after the
parabiosis is established ( 24 ). Using the congenic CD45.1
marker to trace circulating leukocytes in the CD45.2 parabi-
ont, our results reveal that blood-borne dermal langerin + DCs
are constitutively recruited to the dermis and the skin DLNs
in the absence of overt injuries. Similar results are reported in
a companion paper ( 28 ). In this study, the authors show that
in congenic BM chimeric mice, langerin + DCs express higher
levels of CD45 compared with LCs and used the levels of
CD45 expression to identify langerin + DCs, independent of
LCs, in the dermis of naive nontransplanted animals. Alto-
gether, these results strongly support the recruitment of circu-
lating langerin + DCs, independent of LCs, to the dermis in
the steady state.
Congenic BM chimeric mice provide another tool to dis-
tinguish LCs from blood-derived DCs, as LCs remain of host
origin months after transplant despite complete donor-derived
chimerism in the blood. In this model, blood-derived langerin +
DCs were also present in the dermis and accounted for 30%
of total langerin + dermal DCs. Surprisingly, the percentage of
blood-derived langerin + DCs is increased in the skin DLNs,
reaching up to 60% of langerin + DCs. The higher percentage
of donor-derived langerin + DCs in the skin DLNs, compared
with dermal langerin + DCs in congenic BM chimeras, is in-
triguing, and could be partially caused by a minor contribution
of donor-derived LCs, which represent 5 – 10% of total LCs in
areas of skin exposed to fi ght- or grooming-related injuries,
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
Figure 6. Phagocytic processing and migration capacities of blood-derived langerin + DCs. Lethally irradiated F1 (C57BL/6 I-A b ? / ? /BALB/c I-E d+ )
mice were reconstituted with BM cells isolated from donor Langerin-EGFP CD45.1 + C57BL/6 I-A b+ mice. (A, top) Overlaid histograms show YAe staining on
gated host LCs (CD45.2 + CD11c + Langerin-EGFP – ), donor langerin + dermal DCs (CD45.1CD11c + Langerin-EGFP + ), and donor langerin ? dermal DCs
(CD45.2 + CD11c + Langerin-EGFP – ). LCs isolated from F1 (C57BL/6 I-A b+/+ /BALB/c I-E d+ ) mice were used as controls. (A, bottom) YAe staining is shown for
host LC-derived DCs (CD45.2 + CD11c + Langerin-EGFP ? ), donor langerin + DCs (CD45.1 + CD11c + CD8a ? Langerin-EGFP + ), and donor langerin ? DCs (CD45.2 + CD
11c + CD8 ? langerin ? ) in the skin DLNs. Corresponding YAe isotype control (blue) and WT F1-positive control (green) are shown. (B) Mean values ( ± the SD)
of the percentage of YAe + cells among donor langerin + or langerin ? DCs in the dermis and skin DLNs. Representative data from two independent experi-
ments are shown. Each experiment included at least three separately analyzed mice; error bars represent the SD between the results obtained from each
of the three mice. (C) Host LCs in the epidermis, donor langerin + , or langerin ? DCs in the dermis, LC-derived DCs, or donor langerin + or langerin ? DCs in
the skin DLNs were purifi ed by cell sorter and stained on cytospin for YAe and langerin as described in Materials and methods. Insets show YAe isotype
control for each tested DC population. Dashed white lines indicate where cells were spliced from different fi elds to present more cells within a panel.
Bar, 10 ? m.
JEM VOL. 204, December 24, 2007
CCR2 also plays a critical role in the release of BM mono-
cytes into the bloodstream ( 34 ), and mice reconstituted with
CCR2 ? / ? BM have a decreased number of circulating mono-
cytes ( 27 ). Therefore, it is possible that the decrease of CCR2 ? / ?
blood-derived langerin + DCs in the dermis refl ects a reduced
capacity for CCR2 ? / ? monocytes to reach the peripheral
blood and diff erentiate into dermal DCs in the quiescent skin.
These results support the recent findings that monocytes
participate in DC homeostasis in peripheral tissues, but not in
lymphoid organs ( 47 ), and the role of CCR2 in the develop-
ment of nonlymphoid DC populations other than skin is cur-
rently being analyzed in the laboratory.
Consistent with previous studies, our results show that af-
ter systemic depletion of langerin + cells in vivo, the repopula-
tion of dermal and skin DLN langerin + DCs precedes the
repopulation of epidermal LCs ( 18, 23 ). We also demonstrate
that dermal langerin + DCs, independent of LCs, participate in
the repopulation of the langerin + DC pool in the skin DLNs,
whereas the epidermis remains empty of LCs. It is intriguing
that langerin + dermal DCs migrate to the skin DLNs before
seeding an epidermis depleted of LCs, suggesting that blood-
derived langerin + DCs cannot diff erentiate into LCs in vivo.
Another possibility is that the toxin used to deplete langerin +
cells leads to infl ammatory signals that trigger the migration of
blood-derived dermal langerin + DCs to the skin DLNs before
they can reach the epidermis. This is unlikely because we have
shown that circulating LC precursors can seed an infl amed
epidermis and diff erentiate into LCs ( 33 ). In addition, we and
others have not detected signs of skin infl ammation or injuries
after DT injection ([ 18 ] and unpublished data). A more likely
explanation is that in vivo conditional LC ablation with DT
frees available LC niches, but fails to induce epidermal injury
signals, precluding the recruitment of blood-derived precur-
sors to the epidermis. It will be interesting to examine if in-
duction of infl ammatory cytokines and chemokines in the
epidermis after DT treatment accelerates the recruitment of
dermal langerin + DCs to the epidermis. If this is true, these
data, together with our previous data showing that monocytes
are the precursors of LCs in infl amed skin, will support the
hypothesis that monocytes are constitutively recruited to the
dermis where they diff erentiate into langerin + DCs dedicated
to survey the epidermis or the dermal – epidermal junction.
The acquisition of the langerin receptor on skin-infi ltrating
monocytes or a committed DC precursor might represent the
signature of a “ LC-like ” diff erentiation program induced by
environmental factors such as TGF ? , for example ( 48 ). Sup-
porting this hypothesis is our fi nding that dermal langerin + DCs
seem to progressively acquire the LC marker langerin while
losing the monocyte marker CX3CR1, a marker that is also
absent on LCs, but partially expressed by dermal langerin – DCs
( Fig. 4 C ). Whether dermal langerin + and dermal langerin – DCs
represent the progeny of a precursor that follows a separate dif-
ferentiation pathway, with the dermal langerin + DCs being an
intermediate precursor to LCs, is plausible, but diffi cult to test
because of the limitation of an experimental system dealing
with a low number of cells. More information will be needed
We also examined the mechanisms that regulate the re-
cruitment of blood-derived langerin + DCs to the dermis. Our
data suggest that blood-derived langerin + DCs use E- and
P-selectin to enter the skin in the absence of skin injuries. These
data are consistent with a previous study showing that human
CD34 + -derived DCs injected into immunocompromised mice
roll continuously along noninfl amed mouse dermal endo-
thelium in an E- and P-selectin – dependent manner ( 31 ).
Our data also show that CCR2 controls the recruitment of
blood-derived langerin + DCs to the quiescent dermis. Ex-
pression of CCR2 ( 45 ) on circulating leukocytes was shown
to be required for the repopulation of LCs ( 32, 46 ) and dermal
langerin – DCs in infl amed skin ( 27 ). Our results represent the
fi rst indication for a role of CCR2 in cutaneous DC homeo-
stasis in the absence of overt skin infl ammation. However, it
remains possible that the transplant model used to reveal the
role of CCR2 might lead to x ray – induced skin injuries, trig-
gering the release of CCR2 ligands and the recruitment of
circulating DCs that will not be encountered in the steady state.
Figure 7. Origin of langerin + DC in mice. This diagram illustrates a
hypothetical view on cutaneous DC differentiation pathways in mice in
the steady state. Epidermal LCs are maintained by radioresistant hemato-
poietic progenitors that have taken residence in the skin before birth,
whereas the majority of dermal DCs derive from circulating DC precur-
sors. Our results now suggest that circulating blood-derived CCR2 + DCs
are constitutively recruited to the dermis. E- and/or P-selectin direct this
recruitment in transplanted animals, and studies are conducted in the
laboratory to examine if this is also true in nontransplanted mice. In
response to cutaneous factors, CCR2 + DCs differentiate into dermal
langerin + DCs, but might also be able to differentiate into dermal
langerin – CX3CR1 +/ ? DCs. Dermal langerin + DCs transit, but do not accu-
mulate in the dermis, where they capture skin antigens, before emigrating
to the skin DLNs to present skin-derived peptides in the context of MHC
molecules. Both LCs and dermal DCs require CCR7 to migrate to the T
cells area of skin DLNs. In the skin DLNs, dermal langerin + DCs are undis-
tinguishable from LC-derived DCs, and they are characterized as CD8 –
langerin + CD11c lo , but differ from blood-derived CD8 + langerin lo CD11 hi DCs.
Whether CCR2 + blood precursors that give rise to dermal langerin + DCs
and contribute to CD8 – langerin + CD11c lo in the skin DLNs represent a
committed DC precursor or a circulating monocyte is currently under
study in the laboratory.
ORIGIN OF LANGERIN + DENDRITIC CELLS | Ginhoux et al.
were analyzed with FlowJo software (Tree Star, Inc.). Fluorochrome- or
biotin-conjugated monoclonal antibodies specifi c to mouse B220 (RA3-6B2),
CD8a (53 – 6.7), I-A b (AF6-120.1), IA/IE (M5/114.15.2), CD11b (M1/70),
CD11c (N418), CD45 (30F11), CD45.1 (A20), CD45.2 (104), CD115
(AFS98), Gr-1Ly6C (1A8), Gr-1Ly6C/G (RB6-8C5), CD3 (17A2), CD4
(clone L3T4), anti – mouse E ? 52-68 peptide bound to the I-A b /MHC Class II
(YAe), the corresponding isotype controls, and the secondary reagents (allo-
phycocyanin, peridinin chlorophyll protein, and phycoerythrin – indotricarbo-
cyanine – conjugated streptavidin) were purchased either from BD Biosciences
or eBioscience. For YAe staining, naive untreated cells from C57BL/6 mice
that lack I-E molecules were used as negative controls, and cells from C57BL/6
BALB/c F1 mice were used as positive controls. Anti – mouse CD16-32 (BD
Biosciences) and biotinylated mouse IgG2b isotype control (BD Biosciences)
were used to block Fc III/II receptors and as a YAe isotype control antibody,
respectively. F4/80 (A3-1) was purchased from Serotec. Polyclonal antibody
to langerin was purchased from Santa Cruz Biotechnology. Intracellular stain-
ing against langerin was performed with the BD Cytofi x/Cytoperm kit (BD
Biosciences) according to the manufacturer ’ s protocol. For cytospin immuno-
staining, stained cell suspensions were sorted using an inFlux cell sorter
(Cytopeia, Inc.) and achieve 97 – 99% purity.
Transplantation of BM cells. 8-wk-old recipient CD45.2 + or CD45.1 +
C57BL/6 mice were lethally irradiated with 1,200 rads, delivered in 2 doses
of 600 rads each, 3 h apart, and were injected i.v. with 10 6 BM cells obtained
from congenic CD45.1 + or CD45.2 + C57BL/6 adult mice, respectively.
To address the role of CCR2 and CCR6 in dermal DC langerin +/ ? homeostasis,
lethally irradiated CD45.1 + C57BL/6 mice were reconstituted with a 1:1
mixture of WT CD45.1 + BM cells and CD45.2 + BM isolated from WT,
CCR2 KO, or CCR6 KO mutant mice. In all transplanted mice, levels
of blood donor chimerism were analyzed by measuring the percentage of
donor cells among total B220 + B cells, Ly6C/G + CD115 – granulocytes, and
CD115 + monocytes in the blood 3 wk after transplantation.
Preparation of epidermal and dermal cell suspension. Skin cell sus-
pensions were isolated as previously described ( 24 ) and were analyzed by
fl ow cytometry. In brief, mouse ears were split in two parts (dorsal and ven-
tral) and incubated for 60 min in PBS containing 0.5% trypsin with 5 mM
EDTA (Invitrogen) to allow for separation of dermal and epidermal sheets.
Epidermal and dermal sheets were then cut into small pieces and incubated
for 2.5 h in collagenase D (1.6 mg/ml, working activity of 226 U/mg;
Worthington) to obtain homogeneous cell suspension. The analysis of he-
matopoietic cell populations present in skin cell suspensions was assessed by
fl ow cytometry by gating on DAPI – CD45 + cells.
Immunofl uorescence analysis of mouse skin. For staining of frozen sec-
tions, skin samples or skin DLNs were fi rst fi xed for 2 h in PBS 4% parafor-
maldehyde containing 20% sucrose to preserve EGFP fl uorescence and were
rinsed in PBS before freezing in optimum cutting temperature compound.
8- ? m-thick skin sections were prepared and stained with anti-langerin (poly-
clonal goat IgG; Santa Cruz Biotechnology) for 1 h, washed in PBS, and
incubated with secondary reagent donkey anti – goat Cy3 (Jackson Immuno-
research Laboratories). Skin DLN sections were fi rst stained with anti-langerin
(polyclonal goat IgG), followed by donkey anti – goat Cy3, and were stained
after with biotinylated anti-B220 (RA3-6B2; BD Biosciences) revealed with
streptavidin-Cy5 (Vector Laboratories). Slides were mounted with Vecta-
shield containing DAPI (4,6-diamidino-2-phenylindole; Vector Laborato-
ries). Images were acquired with a laser scanning confocal microscope
(TCS-SP; Leica) and analyzed with Image J software (National Institutes
Immunofl uorescence analysis of cytospins. Cytospins were prepared
from 5 – 10,000 sorted cells, air-dried, and fi xed in 3% paraformaldehyde.
Slides were incubated with 0.8 ? g/ml anti-langerin goat polyclonal antibody or
corresponding control (goat IgG; Sigma-Aldrich). For YAe staining, after block-
ing with 5 ? g/ml mouse IgG2b (eBioscience) and 2.5 ? g/ml anti-CD16/32
to compare the nature of the antigen cargo displayed by these
diff erent sources of antigen-presenting cells in the skin DLNs to
assess the immune relevance of each of these DC populations.
Our study also reports a striking diff erence between the ho-
meostasis and traffi cking pattern of LCs and dermal langerin +
DCs. After conditional ablation of langerin + DCs in vivo, we
were surprised to discover that dermal langerin + DCs reappear
in 5 d and start to emigrate to the skin DLNs and repopulate the
langerin + DC pool immediately after, whereas LCs remained
absent from the epidermis for > 2 wk after their elimination.
The constitutive recruitment of blood-derived langerin + DCs
to the dermis, together with their rapid transit through the skin
before reaching the skin DLNs, strongly support their role in
skin immunosurveillance. This role is further supported by our
fi ndings showing that blood-derived dermal langerin + DCs cap-
ture and process skin-derived antigens before emigrating to the
T cell areas of the skin DLNs, where they present skin-derived
peptides in the context of MHC class II molecules. These fi nd-
ings establish that, in addition to LCs and dermal langerin – DCs,
an additional population of antigen-presenting cells may play a
role in skin immunity. The expression of the langerin receptor
on the surface of dermal DCs should allow the sampling of an-
tigen patterns by LCs, but not by dermal langerin – DCs.
Our results reveal a novel pathway of cutaneous DC dif-
ferentiation and homeostasis that add to the complexity of the
DC network system developed to support skin immune re-
sponses ( Fig. 7 ). We identifi ed a population of circulating
langerin + DCs, independent of LCs, that constitutively pa-
trols the dermis to capture and process skin antigens before
emigrating to the skin DLNs, where they present skin-derived
peptides – MHC complexes ( Fig. 7 ). This study uncovers a
previously unappreciated element of skin immunosurveil-
lance that is likely to impact the design of vaccine strategies.
MATERIALS AND METHODS
Mice. BALB/c, C57BL/6 (CD45.2 + ), and congenic C57BL/6 CD45.1 + mice
5 – 8 wk of age were purchased from The Jackson Laboratory. Langerin-EGFP,
Langerin-DTR/EGFP mice were generated as previously described ( 18 ).
CCR2-deficient C57BL/6 ( 45 ) and CCR6-deficient C57BL/6 ( 49 ),
CD62L ? / ? ( 50 ) and CCR7 ? / ? ( 30 ) (a gift from J. Lowe, Cleveland Clinic,
Cleveland, OH), and P/E-selectin – defi cient (P/E ? / ? ) mice ( 51 ) (a gift from
P. Frenette, Mount Sinai School of Medicine, New York, NY) were bred and
maintained at our animal facility (Mount Sinai School of Medicine, New
York, NY). CD45.1 + Langerin-EGFP mice were generated by crossing of
congenic C57BL/6 CD45.1 + with CD45.2 + Langerin-EGFP. C57BL/6
BALB/c F1 and I-A b ? / ? C57BL/6 BALB/c F1 were generated by breeding
BALB/c with C57BL/6 or I-A b ? / ? C57BL/6 (The Jackson Laboratory),
respectively. All mice used for experiments were between 8 and 12 wk of age.
Parabiotic mice were generated as previously described ( 36 ). Pairs of parabiotic
mice consisted of congenic C57BL/6 CD45.1 + mice linked to either C57BL/6
CD45.2 + or Langerin-DTR/EGFP C57BL/6 CD45.2 + mice. Parabiotic mice
were killed ? 2 mo after initiation of parabiosis and subjected to tissue analysis.
To confi rm effi cient blood mixing in parabiotic mice, the percentage of
CD45.1 + and CD45.2 + cells among blood leukocytes was analyzed in each
animal. All animal protocols were approved by the Institutional Committee
on Animal Welfare of the Mount Sinai Medical School.
Flow cytometry and cell sorting. Multiparameter analyses of stained cell
suspensions were made on an LSR II fl ow cytometer (Becton Dickinson) and
JEM VOL. 204, December 24, 2007
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antibodies (BD Biosciences), slides were incubated with 2.5 ? g/ml anti-YAe
biotinylated antibody (eBioscience) and 0.8 ? g/ml anti-langerin goat poly-
clonal antibody or isotype controls. Secondary detection of YAe was achieved
using biotinyl-tyramide amplifi cation (kit NEL700A; Perkin Elmer) combined
with 1 ? g/ml Cy5-conjugated streptavidin (Jackson Immunoresearch Lab-
oratories) and 1 ? g/ml Cy3-conjugated donkey anti – goat (Jackson Immuno-
research Laboratories). GFP fl uorescence was usually not detectable in these
preparations. Images were acquired using an Axiophot microscope at 40 ×
magnifi cation (Carl Zeiss, Inc.).
Online supplemental material. Fig. S1 depicts the gating strategy used in
the dermis and in the skin DLNs to analyze all DC subsets by fl ow cytometry.
The online version of this article is available at http://www.jem.org/cgi/
The authors would like to thank Drs. J. Donovan and Y. Kirkorian for their review of
This work was supported by grants from the National Institutes of Health
(RO1-CA112100), the Leukemia and Lymphoma Society (FG, 3220-08), the
Leukaemia Research Fund Bennett Senior Fellowship (M.P. Collin), and the Amy
Strelzer Manasevit Research program (M. Bogunovic).
The authors have no confl icting fi nancial interests.
Submitted: 14 August 2007
Accepted: 21 November 2007
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