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Supplementary Methods and Data -- INVENTORY
1. Supplemental Methods Mice (partial)
PCR
Flow cytometry
Epidermal sheets
Lymph node/spleen cell suspensions/isolation of splenic dendritic cells
Bone marrow chimeras
BrdU treatment of neonatal mice and subsequent analysis of cell cycle phases
Epidermal skin explant culture
Repopulation assay
Intravital microscopy
Generation of bone marrow derived dendritic cells (BMDCs)
2. Supplemental Figures and corresponding Legends: Figure S1: CD11c-p14del mice specifically lack Langerin+ migratory DCs in the skin draining
LNs. [corresponding to Figure 1]
Figure S2: Bone marrow (BM) transfer and analysis of LC repopulation in the skin and LNs.
[corresponding to Figure 2]
Figure S3: Video (z-stack) of intravital microscopy after reconstitution of LC-depleted Langerin-
DTR mice with LangerinEGFP BM: 7 weeks (online). [corresponding to Figure 2]
Figure S4: Video (z-stack) of intravital microscopy after reconstitution of LC-depleted Langerin-
DTR mice with LangerinEGFP BM: 13 weeks (online). [corresponding to Figure 2]
Figure S5: Loss of LCs in CD11c-p14del mice is not due to increased migration of LCs.
[corresponding to Figure 4]
Figure S6: Gating strategy for cell cycle analysis of LCs and DETCs based on BrdU and 7AAD
incorporation. [corresponding to Figure 5]
Figure S7: Langerin-specific depletion of p14 leads to the recruitment of short-term LCs and
long-term LCs to the inflamed skin. [corresponding to Figure 6]
2
1. Supplemental Methods:
Mice. Wild type C57BL/6 were from Charles River Laboratories, Sulzfeld, Germany.
LangerinEGFP1 and LangerinDTR mice1 were kindly provided by B. Malissen, France. CD11c-
Cre2 and Langerin-Cre3 mice were from co-authors B. Reizis and B. E. Clausen, respectively. Co-
author L.A. Huber contributed p14-flox mice4. Rosa26-tdTomato mice were purchased from the
Jackson Laboratory (Cat. #007914). Mice were bred at the animal facility of the Department of
Dermatology & Venereology and were used until the age of 2 months. All experimental protocols
were approved by the Austrian Federal Ministry of Science and Research and performed
according to institutional guidelines.
PCR. DNA was isolated from the tail of mice using QIAGEN TailLysis Buffer (QIAGEN).
Appropriate primers (Microsynth, Balgach, Switzerland) and analysis of particular genetic loci as
described previously4.
Flow cytometry. All antibody staining steps were performed at 4°C. Nonspecific FcR-mediated
antibody staining was blocked by incubation for 5 min with anti-CD16/32 Ab (2.4G2, in-house
from hybridoma supernatant). The following antibodies were used: anti-langerin-FITC (929.F3,
Dendritics, Lyon, France), anti-MHC class II-FITC (553632), anti-CD86-PE (553692), anti-CD40-
PE (55379) and anti-CD103-PE (557495) (all from BD Biosciences, Vienna, Austria), anti-CD86-
PE (105006, Biolegend, San Diego, USA), anti-CD11c-PE-Cy5 (15-0114-81) and anti-MHC II-
APC (17-5321-81, both from eBioscience, San Diego, USA), APC-coupled Life/Dead Cell stain
kit (L10120, Life Technologies, Carlsbad, USA). Flow cytometry was performed on a BD
Biosciences FACSCalibur or BD Biosciences Canto II with data analysis using FlowJo software
(Tree Star, Olten, Switzerland).
Epidermal sheets. Epidermis was separated from dermis using 0.1M ammoniumthiocyanate
(Merck). The epidermis was peeled off and fixed in acetone or 4% PFA (SAV-LP, Flintsbach,
Germany). Epidermis was stained with the following antibodies: pure or FITC-conjugated anti-
3
langerin (clone 929.F3, Dendritics, Lyon, France), anti-MHC class II (B21.2, hybridoma cells
provided by Dr. Ralph Steinman, Rockefeller University), anti-active caspase-3 (AF835, R&D
Systems), and anti-phospho-Histone H3 (ab5176, Abcam, Cambridge, UK). The following
secondary antibodies were used: Alexa fluor488 goat anti-rabbit IgG or goat anti-rat IgG
(A11034, A11006) as well as Alexa fluor 568 goat anti-rat IgG (A11077), all from Life
Technologies.
Lymph node/spleen cell suspensions/isolation of splenic DCs. Skin draining lymph nodes
and spleens were teased apart and resulting fragments digested (25 min./37°C) with 0.12mg/ml
of DNAse I (Roche) and 0.5mg/ml of collagenase P (Roche). DCs were isolated using CD11c-
specific Microbeads (MACS Cell Separation Reagents, Miltenyi Biotec, Bergisch-Gladbach,
Germany) according to the manufacturer’s guidelines.
Bone marrow chimeras. Recipient mice were lethally irradiated with 10 Gy and subsequently
reconstituted i.v. with 5x106 total bone marrow (BM) cells. For LC repopulation, LangerinDTR
mice were depleted of LCs by intra-peritoneal injection with 500ng of diphtheria toxin
(Calbiochem, Darmstadt, Germany)1 24 hours before lethal irradiation.
BrdU treatment of neonatal mice and subsequent analysis of cell cycle phases. Mice
received two injections of BrdU (Sigma-Aldrich, Vienna, Austria) at 16 and 2 hours prior the
experiment. A volume of 100µl was administered subcutaneously into the abdominal skin (50µg/g
bodyweight). The FITC-BrdU Flow Kit (BD Bioscience, Cat. 559619) was used according to the
manufacturer's guidelines.
Epidermal skin explant culture. Whole body wall skin from neonatal mice at an age of 5 days
was placed epidermal side up onto RPMI1640 (PAA, Pasching, Austria) supplemented with 1.2
units/ml of Dispase II (Roche, Germany). The skin was incubated for 40 min at 37°C. Thereafter,
epidermis and dermis were separated from each other, and the epidermis was subsequently
placed dermal side down onto complete medium.5 The epidermis was cultured for 72 hours at
37°C. Emigrated LCs were collected and analyzed by flow cytometry.
4
Repopulation assay. Dorsal ear skin of anesthetized mice was treated with 25µl of 1% TNCB
(picryl chloride). TNCB was dissolved in acetone:olive oil (4:1). LCs were analyzed on indicated
time points after TNCB application.
Intravital microscopy. Mice were anesthetized and the ear was fixed between two microscope
glass slides. The mouse was placed on the stage and the slides were adjusted with the dorsal
side of the ear facing upwards. Microscopy was performed with a microlens-enhanced Nipkow
disk-based UltraVIEW RS spinning disc unit (Perkin Elmer, Massachusetts, USA), mounted on an
Olympus IX-70 inverted microscope.
Generation of bone marrow-derived DCs (BMDCs). BM was isolated from the hind limbs.
Unfractionated BM cells were cultured in complete medium containing 200ng/ml of GM-CSF (14-
8332-62, eBioscience) for a total of 8 days.
References 1. Kissenpfennig A, Henri S, Dubois B, et al. Dynamics and function of Langerhans cells in vivo: Dermal dendritic cells
colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity. 2005; 22:643–654.
2. Caton, ML, Smith-Raska, MR, Reizis, B. Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in
the spleen. J Exp Med. 2007; 204:1653–1664.
3. Zahner SP, Kel JM, Martina CA, Brouwers-Haspels I, van Roon MA, Clausen BE. Conditional Deletion of TGF-
betaR1 Using Langerin-Cre Mice Results in Langerhans Cell Deficiency and Reduced Contact Hypersensitivity. J
Immunol. 2011;187:5069–5076.
4. Teis D, Taub N, Kurzbauer R, et al. p14-MP1-MEK1 signaling regulates endosomal traffic and cellular proliferation
during tissue homeostasis. J Cell Biol. 2006; 175:861–868.
5. Stoitzner P, Romani N, McLellan AD, Tripp CH, Ebner S. Isolation of Skin Dendritic Cells from Mouse and Man.
Methods Mol Biol. 2010; 595:235–248.
7
Figure S1, corresponding to Figure 1: CD11c-p14del mice specifically lack LCs and
Langerin+ migratory DCs in the skin-draining LN. (A,B) Analysis of common DC subsets in the
skin-draining LNs of 6 week old CD11c-p14del and control mice. LN-resident DCs were subdivided
into CD11c+CD4+CD8neg. DCs, CD11c+CD4neg.CD8+ DCs and CD11c+CD4neg.CD8neg. DCs. pDCs
were characterized as PDCA1+ DCs (pre-gated on CD11c+ DCs). Migratory DCs were subdivided
into langerin+ and langerinneg. subsets. Cells were pre-gated for viable cells. One representative
mouse out of 4 is shown in A; combined data from at least 4 individually analyzed mice per
genotype in B. (C) Immunfluorescence staining of epidermal sheets, derived from an adult (6
weeks) CD11c-p14del and a control mouse. LCs are stained for MHC class II (red), DETCs
(dendritic epidermal T cells) for CD3 (green). CD11c-p14del mice lack virtually all LCs. However, in
both mice the DETC network is fully intact (Scale bar: 100µm). * p<0.05, ** p<0.01, *** p<0.001.
8
Figure S2, corresponding to Figure
2: BM transfer and analysis of LC re-
population in the skin and LNs. (A)
Scheme for LC depletion and
subsequent BM transfer in order to
investigate LC repopulation in the skin.
(B) Intravital microscopy for LC
repopulation in LC-depleted
LangerinDTR mice, 3 and 13 weeks
after reconstitution with 5x106 BM cells
from LangerinEGFP mice. At 3 weeks
only few EGFP+ cells were scattered
across the epidermis of recipient mice
(B, upper panels, arrow heads). At 13
weeks a large number of EGFP+ LCs
could be observed (B, lower panels)
(Scale bar: 50µm). (C) Analysis of skin-
draining LNs of reconstituted mice 20
weeks after BM transfer. LCs were
identified as langerin+/ CD103neg cells.
Donor/host contribution was analysed
based on the percentage of EGFP+ to
EGFPneg cells (in the left and middle
column). One representative mouse for
each BM chimera in C (n=3).
9
Video S3, corresponding to Figure 2: Video (z-stack) of intravital microscopy after
reconstitution of LC depleted Langerin-DTR mice with LangerinEGFP BM: 7 weeks (online).
Video shows repopulating EGFP+ LCs (grey) in the epidermis 7 weeks after BM transfer.
Video S4, corresponding to Figure 2: Video (z-stack) of intravital microscopy after
reconstitution of LC depleted Langerin-DTR mice with LangerinEGFP BM: 13 weeks (online).
Video shows repopulating EGFP+ LCs (grey) in the epidermis 13 weeks after BM transfer.
10
Figure S5, corresponding to Figure 4: Loss of LCs in CD11c-p14del mice is not due to
increased migration of LCs. (A) Skin draining LNs (auricular, brachial and inguinal LNs) from 9-
day old mice were analyzed for numbers of migratory DCs. CD11c-p14del mice have less CD40hi,
migratory DCs (B, left graph), as well as less CD40hilangerin+ DCs (B, right graph) as compared
to control mice. One representative mouse out of 3 is shown in A; combined data from 3
individually analyzed mice per genotype in B. (C) CCR7 expression of LCs emigrated from
epidermal explant cultures, derived from mice of postnatal day 5. Histograms show expression of
CD40 and CCR7 on gated viable MHC II+ LC (Isotype: grey filled; control, dotted line; CD11c-
p14del, black line). CD11c-p14del and control LCs up-regulate CCR7 as shown by comparison of
freshly isolated LCs (D) and emigrated LCs (E). One representative experiment of an epidermal
explant culture at day 3 of culture in C; combined data from 3 individually analyzed mice in D and
E. * p<0.05, ** p<0.01, *** p<0.001.
11
Figure S6, corresponding to Figure 5: Gating strategy for cell cycle analysis of LCs and
DETCs based on BrdU and 7AAD incorporation. (A) Cells were pre-gated according to FSC-
Area and SSC-Area to exclude cell debris, followed by LIVE/DEAD separation and doublet
discrimination based on FSC-Area versus FSC-Width. LCs were identified as MHC II+ cells,
DETCs as CD3+ cells. (B) Example for LCs, subdivided into the three cell cycle phases, resulting
from BrdU versus 7AAD analysis: G1/G0 phase: BrdUneg., 7AADlow, S phase: BrdU+, 7AADlow-high,
G2/M phase: BrdUneg., 7AADhigh.
12
Figure S7, corresponding to Figure 6: Langerin-specific depletion of p14 in Langerin-p14del mice leads to the recruitment of short-term LCs and long-term LCs to the inflamed skin. (A,B) Analysis of the total epidermal LC population (MHC II+ cells) on day 0 (i.e., untreated) as
well as 7, 21 and 35 days after TNCB treatment. The proportions of short-term LCs (MHC
II+langerinneg cells) and long-term LCs (MHC II+langerin+ cells) were determined for each time
point. One representative experiment of each genotype and time point is shown in A; combined
data from at least 4 individually analyzed mice per genotype and time point in B. (C) Analysis of
CD11c expression by MHCII+langerin+ long-term LCs and MHCII+langerinneg short-term LCs
derived form control mice, 7 days after TNCB treatment. DETCs were used as internal negative
control for CD11c expression (histogram). (D) Quantification of CD11c expression by long-term,
short-term LCs and DETCs. One representative experiment out of three is shown in C; combined
data from 3 individually analyzed mice per genotype in D. * p<0.05, ** p<0.01, *** p<0.001.