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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]

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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-

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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.

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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.

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2. Supplemental Figures and corresponding Legends:

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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.

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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).

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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.

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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.

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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.

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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.