Epidermal RANKL controls regulatory T-cell numbersvia activation of dendritic cellsKarin Loser1,2, Annette Mehling1, Stefanie Loeser3, Jenny Apelt1, Annegret Kuhn4, Stephan Grabbe5,Thomas Schwarz6, Josef M Penninger3 & Stefan Beissert1,2

Regulatory CD4+CD25+ T cells are important in suppressing immune responses. The requirements for the maintenance of

peripheral CD4+CD25+ T cells remain incompletely understood. Receptor activator of NF-jB (RANK) and its ligand (RANKL; also

known as CD254, OPGL and TRANCE) are key regulators of bone remodeling, mammary gland formation, lymph node development

and T-cell/dendritic cell communication. Here we report that RANKL is expressed in keratinocytes of the inflamed skin. RANKL

overexpression in keratinocytes resulted in functional alterations of epidermal dendritic cells and systemic increases of regulatory

CD4+CD25+ T cells. Thus, epidermal RANKL expression can change dendritic cell functions to maintain the number of peripheral

CD4+CD25+ regulatory T cells. Epidermal RANKL mediated ultraviolet-induced immunosuppression and overexpression of

epidermal RANKL suppressed allergic contact hypersensitivity responses and the development of systemic autoimmunity.

Therefore, environmental stimuli at the skin can rewire the local and systemic immune system by means of RANKL.

Several mechanisms are believed to contribute to the maintenance ofperipheral tolerance to autoreactive T cells that have escaped negativeselection in the thymus. These mechanisms include suppression ofautoaggressive T cells by CD4+CD25+ regulatory T cells, and this effectseems to be increasingly important for the avoidance of autoimmunityin mice and humans1–3. CD4+CD25+ T cells seem to develop as aunique lineage controlled by expression of Foxp3 (refs. 4–6).

The molecular mechanisms involved in the peripheral homeostasisof CD4+CD25+ T cells are incompletely understood. Although anergic,CD4+CD25+ T cells undergo rapid expansion in lymphopenic hostmice. Studies in Il2–/– and Il2rb–/– mice suggest that interleukin-2 (IL-2)is important for the peripheral survival of CD4+CD25+ T cells7,8, andcostimulatory signals, in particular those mediated through an interac-tion between B7 and cytotoxic T lymphocyte–associated antigen 4(CTLA-4)/CD28, also seem important for driving CD4+CD25+

T cell expansion9. Costimulatory molecules are also required onantigen-presenting cells (APC) for effective T-cell priming, pointingto a possible role for APC in modulating regulatory T cells. Indeed,dendritic cells (DCs) can induce CD4+CD25+ T-cell proliferation andexpansion10 as well as their development from CD4+CD25– T cells11.These findings show that DCs are able to functionally regulateCD4+CD25+ T cells, but the underlying molecular mechanisms andthe DC subtypes involved are unclear. Members of the tumor necrosisfactor (TNF) family, such as CD40L, are important regulators of DCfunction. The CD40L-mediated activation of epidermal Langerhanscells (LC) is known to break tolerance to self, resulting in the develop-ment of severe systemic autoimmunity12. The receptor-ligand pair most

closely related to CD40-CD40L among the TNF family is the receptorRANK and its ligand RANKL (refs. 13,14). Here we show thatRANKL is expressed in keratinocytes of the skin and, by regulatingthe function of epidermal DCs, is crucial for the peripheral homeostasisof regulatory T cells.

RESULTS

Cutaneous RANKL expression induces immunosuppression

RANKL is highly expressed in osteoblast/stromal cells and activatedT cells. RANKL expression can be upregulated by bone resorbingfactors such as vitamin D3 and inflammatory cytokines15. Moreover,RANKL expression can be upregulated on mammary gland epithelialcells in response to pregnancy hormones, suggesting that RANKLexpression could be inducible in other epithelial tissues16. We there-fore analyzed whether RANKL is induced in keratinocytes in the skinfollowing inflammation. Whereas normal skin keratinocytes did notexpress RANKL, inflammation of the skin due to ultraviolet exposureor infection resulted in RANKL expression (Fig. 1a). Moreover, wefound RANKL expression in the keratinocyte cell line PAM212(Fig. 1a). To investigate RANKL expression in human skin, weobtained biopsies from healthy volunteers and from individuals withpsoriasis and cutaneous lupus erythematosus (CLE), and double-stained these using RANKL and cytokeratin antibodies. We detectedlow levels of RANKL expression in keratinocytes of healthy humanskin (Fig. 1b). In psoriatic lesions, we found strong RANKL expres-sion in keratinocytes of all epidermal layers; in inflammatoryCLE lesions, however, we detected no RANKL expression (Fig. 1b).

Received 3 March; accepted 6 November; published online 3 December 2006; doi:10.1038/nm1518

1Department of Dermatology, and 2Interdisciplinary Center of Clinical Research (IZKF), University of Munster, D-48149 Munster, Germany. 3Institute of MolecularBiotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria. 4Division for Immunogenetics, Tumor Immunology Program, German Cancer ResearchCenter, D-69120 Heidelberg, Germany. 5Department of Dermatology, University of Essen, D-45122 Essen, Germany. 6Department of Dermatology, University of Kiel,D-24105 Kiel, Germany. Correspondence should be addressed to S.B. (beisser@uni-muenster.de).

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Moreover, ultraviolet exposure induced RANKL expression in mouseskin (Fig. 1b). These data show that RANKL expression is upregulatedin keratinocytes under certain inflammatory conditions and followingexposure to environmental stimuli.

To investigate the potential role of RANKL signaling in cutaneousimmune responses, we generated transgenic (Tg) mice overexpressingfull-length mouse RANKL under transcriptional control of thekeratin-14 (K14) promoter (Fig. 1c). As RANKL can be cleaved intoa soluble form13–15, we analyzed whether overexpression of RANKLwould result in increased systemic RANKL levels. However, noincreased serum concentrations of RANKL were found (Fig. 1d).

We next investigated whether RANKL overexpression could affectinflammatory cutaneous contact hypersensitivity (CHS) responses.K14-RANKL Tg mice exhibited a substantially decreased CHSresponse (Fig. 1e). This finding is in contrast to previous reportsthat overexpression of CD40L, the closest RANKL homolog amongthe TNF superfamily13,14, triggers immunostimulatory effects12. Nota-bly, CHS responses were enhanced in both littermate and K14-RANKLTg mice when RANKL function was blocked by the injection ofRANK-Fc (Fig. 1e). These data show that RANKL overexpression inkeratinocytes results in inhibition of CHS.

Epidermal RANKL increases the number of regulatory T cells

Because CHS responses are controlled by T cells17, we analyzed T-cellsubpopulations in K14-RANKL Tg mice. These mice showed a 2- to3-fold increase in the number of splenic and lymph node CD4+CD25+

regulatory T cells expressing the transcription factor Foxp3 (Fig. 2a,b).This increased number of CD4+CD25+ regulatory T cells in theK14-RANKL Tg mice was dependent on RANKL-mediated signaling,as blockade of this pathway by RANK-Fc reduced the number ofCD4+CD25+ T cells to normal limits. Furthermore, Tnfsf11–/–

(Rankl–/–) mice had markedly reduced numbers of splenicCD4+CD25+ T cells compared to controls (Fig. 2a). Because costi-

mulatory molecules can induce the production of IL-2, which isinvolved in regulatory T-cell maintenance7,8, we analyzed whetherelevated IL-2 might be responsible for the increased number ofregulatory T cells in K14-RANKL Tg mice. Whereas neutralizing IL-2 in vivo by systemic administration of an antibody to IL-2 (anti–IL-2)reduced the number of Foxp3+CD25+ T cells in wild-type mice, thesame treatment did not reduce the number of regulatory T cells inK14-RANKL Tg mice. This suggested that the expansion of regulatoryT cells in K14-RANKL Tg mice was not mediated by IL-2.

CD4+CD25+ T cells develop in the thymus, and K14 expressionhas also been described in medullary thymic epithelial cells18,19. InK14-RANKL Tg and littermate mice, we detected a similar number ofthymic CD4+CD25+ T cells (data not shown). Whereas splenicCD4+CD25+ T cells were reduced in Tnfsf11–/– mice, these mice hada normal number of thymic CD4+CD25+ T cells (wild type, 2.65 ±0.19% of total thymocytes, n ¼ 10; Tnfsf11+/–, 2.31 ± 0.15%, n ¼ 5;Tnfsf11–/–, 2.91 ± 0.1%, n¼ 5). Nonetheless, it is possible that RANKLexpression on thymic epithelial cells was responsible for the expansionof CD4+CD25+ T cells. To test this, we performed thymectomyfollowed by thymus transplantation (Fig. 2a). After transplantation,littermates with a thymus from K14-RANKL Tg mice developed anormal number of CD4+CD25+ T cells. Again, an increased numberof CD4+CD25+ T cells was present in K14-RANKL Tg mice graftedwith a wild-type thymus. Thus, RANK-RANKL interactions seem tobe relevant for the maintenance and/or peripheral expansion, ratherthan the thymic development, of CD4+CD25+ regulatory T cells.

We next examined whether the increased CD4+CD25+ populationin K14-RANKL Tg mice expresses markers and exhibits functionalproperties that are characteristic of regulatory T cells. CD4+CD25+

T cells from K14-RANKL Tg mice did indeed express prototypicsurface markers such as CD45RBlo, neuropilin-1 (Nrp-1), intracellularCTLA-4 and integrin-aE (CD103) (Fig. 2c and refs. 18,20–23).CD4+CD25+ T cells from K14-RANKL Tg mice did not proliferate

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Figure 1 Epidermal RANKL overexpression suppresses cutaneous contact hypersensitivity (CHS)

responses. (a) RANKL is expressed in inflamed skin. RT-PCR analysis of untreated (‘WT skin’), UVB-

irradiated and Herpes simplex virus type 1 (HSV)-infected skin27 from wild-type (WT) mice; inflamed

skin from CD40L transgenic mice (‘Inflammatory skin’); and stimulated and unstimulated PAM212

keratinocytes (‘PAM212’ and ‘PAM212 + IFN-g’). (b) RANKL expression in mouse and human

keratinocytes. Immunofluorescence stainings of skin using antibodies to cytokeratin and RANKL. Scalebar, 50 mm. (c) K14-RANKL Tg expression in basal keratinocytes. Immunohistochemical staining of ear

skin using an antibody to mouse RANKL. Arrows indicate basal keratinocytes. Scale bar, 50 mm.

(d) RANKL concentration in the serum of wild-type and K14-RANKL Tg mice. RANKL levels were

analyzed by ELISA. Data represent mean ± s.d. from five mice for each group. (e) Reduced contact

hypersensitivity responses in K14-RANKL Tg mice. Mice were sensitized (‘Sensi’) with 2,4-

dinitrofluorobenzene (DNFB) and challenged (‘Chall’) with either DNFB or oxazolone (OXA). Groups of

mice were injected with RANK-Fc before sensitization. Ear swelling reaction as a measure of CHS responses was assessed 48 h after challenge. Data are

shown as mean ear swelling ± s.d. and are representative of 15 mice in three independent experiments. *P o 0.05 (Student’s t-test), CHS responses in

wild-type versus K14-RANKL Tg mice. **P o 0.05, CHS responses in K14-RANKL Tg without RANK-Fc versus K14-RANKL Tg with RANK-Fc.

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upon T-cell receptor (TCR) stimulation, and this anergy couldbe overcome by adding IL-2 (Fig. 2d). Regulatory T cells fromboth littermate and K14-RANKL Tg mice produced IL-10 uponstimulation (Fig. 2d).

In humans and mice, CD4+CD25+ regulatory T cells play animportant role in immune tolerance by suppressing self-reactiveT cells1–3,18. We found that upon TCR stimulation, the CD4+CD25+

T cells from K14-RANKL Tg and littermate mice suppressed theproliferation of wild-type CD4+CD25– T cells to a similar extent(Fig. 2e). RANKL expression in the skin did not change the sensitivityof CD4+CD25– T cells to suppression by regulatory T cells, andsuppressor function was contact dependent in vitro (data notshown). Thus, expression of RANKL in keratinocytes resulted in thesystemic expansion of CD4+CD25+ T cells that show phenotypic andfunctional characteristics of regulatory T cells.

Functional alterations of RANKL-activated dendritic cells

RANKL overexpression in the epidermis did not change systemiclevels of soluble RANKL (Fig. 1d), and expression of the RANKLreceptor RANK was not detectable on CD4+CD25+ T cells (data notshown). Besides macrophages and osteoclasts, dendritic-cell subsetsalso constitutively express the RANKL receptor RANK (refs. 13,15,24).Therefore, we analyzed whether LCs, the resident DCs in the epider-mis, express RANK. Indeed we found that epidermal LCs expressRANK protein, as determined by immunofluorescence (Fig. 3a). Wetherefore speculated that enhanced RANK-RANKL signaling occursbetween RANK-expressing LCs and RANKL-expressing keratinocytes.

Under steady-state conditions, LCs continuously migrate from theskin to the draining lymph nodes and have an important role in the

induction of antigen-specific T-cell activation25,26. We found normalnumbers and distributions of epidermal LCs in K14-RANKL Tg mice(Fig. 3a). We also detected similar numbers of FITC+/langerin+ LCs inskin-draining lymph nodes of wild-type and K14-RANKL Tg mice(1.15 ± 0.33% and 1.60 ± 1.07%, respectively, of total lymph nodecells) after epicutaneous FITC application. These results indicate thatRANKL overexpression has no apparent effect on the numbers,distribution or migratory behavior of LCs.

We next surmised that RANKL expression in keratinocytes mightchange the function of LCs, which could result in the peripheralexpansion of CD4+CD25+ T cells. To directly investigate if DCs fromK14-RANKL Tg mice can expand CD4+CD25+ T cells, we addedCD11c+ DCs from peripheral skin-draining lymph nodes of wild-typeand K14-RANKL Tg mice to CD4+CD25+ T cells. Notably, DCs fromK14-RANKL Tg mice induced markedly enhanced proliferation ofallogeneic CD4+CD25+ T cells (Fig. 3b). Similar to lymph node DCs,epidermal LCs from K14-RANKL Tg skin exhibited an increasedcapacity to induce proliferation of allogeneic CD4+CD25+ T cells.These results show that lymph node–derived DCs and epidermal LCsfrom K14-RANKL Tg mice can induce increased proliferation ofCD4+CD25+ T cells.

To test if LCs are indeed involved in regulating the numbers ofperipheral CD4+CD25+ T cells in vivo, epidermal LCs were depletedfrom mouse epidermis by topical treatment with mometason fuorate(Fig. 3c, Supplementary Fig. 1 online and ref. 27). LC depletioninduced a substantial (up to 70%) reduction of lymph nodeCD4+CD25+ T cells in control littermates (Fig. 3d,e). We alsoobserved this reduction in the number of CD4+CD25+ T cells inK14-RANKL Tg mice following LC depletion, which resulted in

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Figure 2 Epidermal RANKL controls the number of peripheral

CD4+CD25+ regulatory T cells. (a) Cutaneous RANKL expression

induced the expansion of CD4+CD25+ T cells. Flow cytometric analyses

of splenic T cells from wild-type mice (n ¼ 70), K14-RANKL Tg

mice (n ¼ 50), K14-RANKL Tg mice treated with RANK-Fc (n ¼ 10)

or control-Fc (n ¼ 5), Tnfsf11–/– mice (n ¼ 5), thymectomizedK14-RANKL Tg mice grafted with a wild-type thymus (n ¼ 8), and

thymectomized wild-type mice grafted with thymic tissue from

K14-RANKL Tg mice (n ¼ 8). Representative dot plots are shown.

(b) Flow cytometric analyses of lymph node T cells from wild-type mice

(n ¼ 12), K14-RANKL Tg mice (n ¼ 12), wild-type mice treated with

anti–IL-2 (n ¼ 4), and K14-RANKL Tg mice treated with anti–IL-2 (n ¼ 4). Representative dot plots for gated CD4+ T cells are shown. (c) CD4+CD25+

T cells from K14-RANKL Tg mice showed surface marker expression characteristic of regulatory T cells. CD4+CD25– (dark gray lines) and CD4+CD25+

T cells (black lines) were analyzed by flow cytometry. CTLA-4 staining was performed after cell permeabilization. Isotype controls are shown in light gray.

(d) CD4+CD25+ T cells were anergic (left) and secreted IL-10 (right). CD4+CD25– and CD4+CD25+ T cells were stimulated with antibodies to CD3 (anti-CD3)

and to CD28 (anti-CD28) with or without recombinant mouse IL-2. For IL-10 production, stimulated CD4+CD25– and CD4+CD25+ T cells (5 � 104) were

analyzed. Data shown are from one experiment (three were conducted). (e) CD4+CD25+ T cells from K14-RANKL Tg mice were immunosuppressive. Wild-

type CD4+CD25– T cells (2 � 105) were cocultured with anti-CD3 and anti-CD28, and with CD4+CD25+ T cells from wild-type or K14-RANKL Tg mice.

Mean values of 3H-thymidine uptake ± s.d. are shown (three independent experiments).

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similar numbers of these cells in K14-RANKL Tg mice and littermates.These findings indicate that epidermal LCs can maintain peripheralnumbers of regulatory CD4+CD25+ T cells.

Next, we analyzed the expression of cell surface markers onepidermal LCs in order to investigate potential differences in LCphenotypes. LCs from K14-RANKL Tg mice showed increased expres-sion of CD205 (DEC205) and CD86 compared to controls (Fig. 4a).CD205 expression has been previously associated with DC-mediatedinduction of CD4+CD25+ regulatory T cells28, and CD86 hasbeen implicated in protection from spontaneous autoimmunity29.Among LCs emigrating from K14-RANKL Tg epidermal sheets(langerin+ cells: 70 ± 18% in the wild type, 75 ± 12% in K14-RANKL Tg; CD3+ cells: 7 ± 2% in the wild type, 5 ± 1% in K14-RANKL Tg) fewer apoptotic cells were detected compared to LCs fromcontrol epidermis, suggesting that RANK-RANKL signaling prolongsthe longevity of LCs (Fig. 4b). LCs from the skin of RANKL Tg miceand littermates also differed in their spontaneous cytokine secretionprofiles for TNF-a, IL-6 and IL-10 (Fig. 4c). Thus, although LCs shownormal distribution and normal expression of major histocompat-ibility complex (MHC) class II, RANK and langerin, and are present innormal numbers in the skin of K14-RANKL Tg mice, exposure of LCsto epidermal RANKL results in LCs that are less susceptible toapoptosis, and that show increased cytokine production and alteredexpression of surface receptors known to be associated with immu-nosuppressive DC functions. Notably, LCs from K14-RANKL Tg mice

have an enhanced capacity to expand CD4+CD25+ regulatory T cellsin vitro and in vivo.

To test whether RANKL-stimulated DCs can induce regulatoryT cells in vivo, DCs were generated, exposed to RANKL, ovalbumin(OVA) pulsed, and injected into TCR transgenic OT-2 mice. Afterthree injections, OVA-specific CD4+ T cells showed increased Foxp3expression (Fig. 4d). Additionally, OVA-specific CD4+CD25+ T cellsisolated from inguinal lymph nodes of OT-2 mice injected withRANKL-stimulated DCs were anergic and suppressive (Fig. 4e). Wenext investigated whether TNF-a might also influence the RANKL-mediated expansion of CD4+CD25+ T cells in K14-RANKL Tg mice,as TNF-a can modulate the number of regulatory T cells30. Notably,OVA-pulsed and RANKL-stimulated DCs from Tnf�/� mice did notinduce CD4+CD25+ regulatory T cells in vivo, indicating that TNF-aproduced by RANKL-stimulated DCs might be an important med-iator of regulatory T-cell expansion (Fig. 4d).

Cutaneous RANKL expression reduces systemic autoimmunity

Next we determined whether CD4+CD25+ T cells from K14-RANKLTg mice could also suppress the development of systemic autoimmu-nity induced by epidermal CD40L overexpression12—that is, whetherRANKL expression in keratinocytes can over-ride this action ofCD40L. We therefore crossed autoimmune-prone CD40L Tg withK14-RANKL Tg mice to obtain double-mutant mice. CD40L single-Tgmice develop a systemic autoimmune disease, manifested by

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Figure 3 Epidermal Langerhans cells control systemic homeostasis of CD4+CD25+

regulatory T cells. (a) K14-RANKL Tg mice had normal numbers of epidermal LCs

expressing RANK, langerin and MHC class II. Epidermal sheets of wild-type and K14-

RANKL Tg mice were stained with antibodies to RANK, langerin and MHC class II.

Merged langerin+ and RANK+ images are shown in yellow. Scale bar, 25 mm.

(b) Lymph node DCs and epidermal LCs from K14-RANKL Tg mice had an increased

capacity to induce proliferation of CD4+CD25+ T cells. DCs from skin-draining lymph

nodes, and epidermal LC of wild-type and K14-RANKL Tg mice were incubated withCFSE-labeled CD4+CD25+ and CD4+CD25– T cells in 1:2 ratio of T cells to APC. Data represent one experiment (of three conducted). (c) Depletion of LC in

K14-RANKL Tg and wild-type mice following topical treatment with mometason fuorate. Ear sheets of wild-type and K14-RANKL Tg mice were stained with

antibodies to MHC class II and langerin. Merged images are shown. Scale bar, 25 mm. (d) LC depletion reduced the number of peripheral CD4+CD25+

T cells in wild-type and K14-RANKL Tg mice. After topical treatment with mometason fuorate, lymph node and spleen cells were analyzed by flow cytometry

for frequencies of CD4+CD25+ T cells. Representative dot plots are shown. (e) Percent reduction in CD4+CD25+ T-cell numbers in treated versus untreated

mice (n ¼ 5 per group). Data represent one experiment (of three conducted). *P o 0.05 (Student’s t-test).

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scleroderma-like dermatitis, nephritis or proteinuria, and antinuclearantibodies12. However, K14-RANKL/CD40L double-Tg mice showeda markedly reduced and delayed onset of autoimmune dermatitis(Fig. 5a). We were surprised to find that all manifestations ofautoimmunity in CD40L Tg mice (the development of dermatitis,cytotoxic CD8+ T cells, antinuclear antibodies, nephritis and protei-nuria) were inhibited in K14-RANKL/CD40L double-Tg mice(Fig. 5a–c and data not shown). Treatment of double-Tg mice withRANK-Fc to block RANK-RANKL signaling reversed the protectiveeffect of epidermal RANKL expression (Fig. 5a). Disease developmentcorrelated with the number of peripheral regulatory T cells, as double-

mutant and K14-RANKL Tg mice had higher numbers of these cellsthan CD40L single-Tg mice (CD4+CD25+ cells as a percent of totallymph node CD4+ T cells: 9.81 ± 0.84%, 19.22 ± 0.77% and 13.88 ±1.15% in CD40L Tg, K14-RANKL Tg and K14-RANKL/CD40Ldouble-Tg, respectively; n ¼ 8 per group; P o 0.05, CD40L Tg versusK14-RANKL/CD40 double-Tg). Moreover, we detected increasedexpression of Foxp3 and Nrp-1 in CD4+ T cells from lymph nodesof CD40L Tg/K14-RANKL double-Tg mice compared to CD40L Tgsingle mutants (Fig. 5d). These data show that RANKL expression inthe skin can suppress local cutaneous hyperallergic responses as well asCD40L-driven systemic autoimmunity.

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Figure 4 Phenotypic and functional differences between Langerhans cells from wild-type andK14-RANKL Tg mice. (a) CD205 and CD86 expression on epidermal LCs. LC from wild-type

(gray) and K14-RANKL Tg (black) epidermis were analyzed by flow cytometry. (b) LCs from wild-

type (gray) and K14-RANKL Tg (black) epidermis were analyzed for spontaneous cell death using

propidium iodide and annexin V staining. (c) Enhanced spontaneous cytokine production by LCs

from K14-RANKL Tg epidermis. (d) RANKL-stimulated bone marrow–derived DCs induced

regulatory T cells in vivo. DCs (3 � 106) from wild-type or Tnf–/– mice were RANKL activated,

OVA pulsed, and injected subcutaneously into OT-2 Tg mice. Cells were gated for Va2+CD4+, and

one representative dot plot for each group is shown. (e) CD4+CD25+ T cells induced by RANKL-

stimulated, OVA-pulsed DCs were anergic and suppressive. Suppression assays were performed by

coculture of CD4+CD25– T cells (1 � 105) from naive OT-2 Tg mice with anti-CD3 and anti-CD28 in the absence or presence of CD4+CD25+ T cells (1 �105) from OT-2 mice injected with differentially stimulated DCs. Mean values of 3H-thymidine uptake ± s.d. are shown from one representative experiment

(three were conducted). *P o 0.05 (Student’s t-test).

CD40L TgCD40L Tg/K14-RANKL TgCD40L Tg/K14-RANKL Tg + RANK-Fc

*

14013012011010000

20

40

60

80

100

Days

Start of RANK-Fc

Dis

ease

dev

elop

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t (%

)

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ANKL Tg

CD40L T

g

CD40L T

g/

K14-R

ANKL Tg

K14-RANKL Tg CD40L TgCD40L Tg/

K14-RANKL Tg

K14-RANKL TgCD40L TgCD40L Tg/K14-RANKL Tg

5

4

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2

1

0

*

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tein

con

cent

ratio

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Nrp-1

Cou

nts

Cou

nts

Foxp3

a b

d

c

Figure 5 Epidermal RANKL suppressed CD40L-induced systemic autoimmunity.

(a) CD40L Tg mice were crossed with K14-RANKL Tg mice and the onset of autoimmune

dermatitis was determined in the different experimental groups. Left, treatment of

CD40L/K14-RANKL double-Tg mice with RANK-Fc abrogated the in vivo epidermal

RANKL effect. Data were obtained from 10 mice for each group. *P o 0.05 (log-rank

test). Right, H&E staining of skin tissue. Scale bar, 50 mm. (b) Epidermal RANKL suppressed the development of antinuclear antibodies. Indirect

fluorescent staining of HEp-2 cells incubated with serum from K14-RANKL Tg, CD40L Tg and CD40L/K14-RANKL double-Tg mice (serum dilution 1:80).

Scale bar, 50 mm. (c) Reduced proteinuria and rescued renal function by epidermal RANKL overexpression in double-mutant mice. Urine was analyzed from

autoimmunity-prone CD40L Tg (n ¼ 10), K14-RANKL Tg (n ¼ 10) and CD40L Tg/K14-RANKL double-Tg (n ¼ 10) mice. Protein concentration was

quantified using Bradford reagent. *P o 0.05 (Student’s t-test). (d) Increased expression of Foxp3 and Nrp-1 in CD4+ T cells from CD40L/K14-RANKL

double-Tg mice compared to CD40L Tg mice. Cells from cervical lymph nodes were analyzed by flow cytometry and gated for CD4.

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RANKL mediates ultraviolet-induced immunosuppression

The skin is an organ where interaction with the environmentfrequently stimulates the immune system. However, it has longbeen known that ultraviolet exposure can result in immuno-suppression. Moreover, epicutaneous immunization with autoanti-genic peptides prevents experimental allergic encephalitis31, andphenotypically immature LCs, known to trigger regulatory T cells,chronically drain from inflamed skin to local lymph nodes in mice andhumans32,33. Thus, the quality of the inflammation in the skin,possibly manifest as the upregulation of certain molecules on kerati-nocytes, seems to dictate the outcome of the immune response. Thecritical molecules for skin-regulated immune homeostasis have beenelusive. Because ultraviolet irradiation upregulates RANKL as well asCD4+Foxp3+ regulatory T cells in the skin (Fig. 1a,b; Fig. 6a), wespeculated that cutaneous RANKL might be the physiologic missinglink that couples ultraviolet radiation to immunosuppression. Wetested this hypothesis in a previously established model of ultraviolet-mediated suppression of CHS responses34. Notably, injections ofRANK-Fc into irradiated mice resulted in protection from ultravio-let-induced immunosuppression, suggesting that RANK-RANKLinteractions mediate the ultraviolet effects (Supplementary Fig. 2online). To definitively demonstrate that ultraviolet-induced immu-nosuppression is mediated by means of cutaneous RANKL, mice weretransplanted with either wild-type skin or Tnfsf11–/– skin. Aftertransplantation, mice were ultraviolet irradiated as well as sensitizedvia the grafted skin tissue. Mice transplanted with Tnfsf11–/–, butnot wild-type, skin were protected from ultraviolet-inducedimmunosuppression, as indicated by normal CHS responses (Supple-mentary Fig. 2). In parallel with the upregulation of epidermalRANKL expression, ultraviolet irradiation induced the expansionof CD4+CD25+ T cells in mouse skin-draining lymph nodes(Fig. 6b) that exhibited suppressor functions (data not shown).These findings show that ultraviolet irradiation can upregulatecutaneous RANKL expression and, notably, that RANK mediatesultraviolet-induced immunosuppression.

DISCUSSION

RANKL and RANK are essential regulators of osteoclast differentiationand control the formation of a lactating mammary gland duringpregnancy15,16,35. In addition, RANKL expression is inducedon activated T cells, and RANK expression can be found on DCs

(refs. 14,15). However, the core function ofRANKL/RANK in the immune system is stillnot well understood. For instance, it has beenreported that RANKL might be important foractivating intestinal DCs, and inhibition ofRANKL-RANK results in reduced colitis36.On the other hand, RANK-Fc treatment canexacerbate disease in an inflammation-mediated Tg model for diabetes and decreasesthe numbers of CD4+CD25+ regulatoryT cells in pancreas-associated tissue37. Ourdata demonstrate that RANKL expression isinducible on keratinocytes and that this is amolecular pathway that couples the epidermisto local and systemic immunosuppression.Notably, one of the strongest inducers ofRANKL expression is vitamin D3, which isgenerated in the skin and has been longknown to be immunosuppressive15. Forinstance, topical vitamin D3 derivatives are

successfully used to treat psoriasis38. Moreover, dexamethasone andvitamin D3 treatment of T cells can induce regulatory T cells39.Whether (similar to UV-mediated immunosuppression) the immu-noregulatory effects of vitamin D3 are mediated via RANKL remainsto be determined.

It has been proposed that the increasing incidence of allergies andautoimmune diseases in recent years might be due to altered exposureto environmental pathogens, as such diseases occur more frequently incountries with limited sun exposure40. Vitamin D3 and ultravioletradiation (from exposure to sunlight) are two of the strongest inducersof RANKL; our data provide a molecular explanation for thesephenomena and point to a previously underestimated role of cuta-neous DCs in the suppression of immune responses. Of note, we didnot detect RANKL expression in the epidermis of patients with CLE(Fig. 1b), an autoimmune disorder triggered by sun exposure41,further pointing to a role of cutaneous RANKL in the suppressionof autoimmunity. Our data indicate that expression of RANKL in theepidermis provides a critical molecular pathway for the generation ofregulatory mechanisms that control self-reactive T-cell responses.RANKL expressed on inflamed or activated keratinocytes seems torewire local LCs that then have the capacity to regulate the number ofperipheral regulatory T cells. Local induction of RANKL/RANK in theskin could be used as a new therapeutic approach for allergies as wellas for systemic autoimmunity through increasing regulatory T cellswhile avoiding systemic side effects.

METHODSMice. K14-RANKL Tg mice were on a C57BL/6/C3H-HeN background. CD40L

Tg and Tnfsf11–/– mice have been described previously12,16,42. All mice were

housed under specific pathogen-free conditions and all experiments were

performed according to institutional regulations.

Reverse-transcription PCR. The shaved backs of the mice were irradiated with

800 mJ/cm2 UVB or epicutaneously infected with Herpes simplex virus, as

described27. Subsequently, we performed RNA extraction, cDNA synthesis and

RT-PCR43,44 using the following primers: RANKL forward, 5¢-TCGC

TCTGTTCCTGTACTTTCG-3¢ and RANKL reverse, 5¢-GTAGGTACGCTTC

CCGATGT-3¢; b-actin forward, 5¢-GTGGGGCGCCCCAGGCACCA-3¢ and

b-actin reverse, 5¢-CTCCTTAATGTCACGCACGATTTC-3¢.

Immunohistochemistry and immunofluorescence. Immunohistochemistry

was performed on cryostat sections of mouse skin and skin from different

patients or epidermal sheets, according to standard methods12,27,43,44. We

+ RANK-Fc

CD25

CD

4

b

UV (1 mJ/cm2)+ RANK-Fc

WT

K14-RANKL Tg

a WT +UV (1 mJ/cm2)

K14-RANKL Tg +UV (1 mJ/cm2)

UV (0.1 mJ/cm2) UV (1 mJ/cm2)

K14-RANKL TgUV (0.1 mJ/cm2)

WT

18.3 10.4 9.5

4.3 4.8 25.5

Figure 6 RANKL mediates ultraviolet-induced immunosuppression. (a) CD4+Foxp3+ regulatory T cells

were present in the skin. Skin biopsies of naive or ultraviolet-irradiated wild-type and K14-RANKL Tg

mice were stained with antibodies to CD4 and Foxp3. Red, CD4; green, Foxp3. Merged images are

shown in yellow. Scale bar, 50 mm. (b) CD4+CD25+ T cells in skin-draining lymph nodes of ultraviolet-

irradiated and/or RANK-Fc–treated mice. Mice were treated with indicated UVB doses on 4 consecutive

days and T cells were analyzed by flow cytometry on day 5. One representative dot plot for each

group is shown.

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used antibodies to cytokeratin, RANK, RANKL (R&D Systems), Foxp3

(eBioscience), CD4 (eBioscience), MHC class II (BD Pharmingen) and

CD207, or an isotype control antibody (eBioscience). Subsequently, the sections

were incubated with a horseradish peroxidase (HRP)-coupled, Oregon Green–

or Texas Red–labeled secondary antibody (Molecular Probes). For removal of

epidermal LCs, mice were topically treated with mometason fuorate (Ecural,

Essex Pharma) on 4 consecutive days per week for 4 weeks. One week after the

last mometason fuorate treatment, spleens and regional lymph nodes were

removed and single-cell suspensions prepared for flow cytometry.

RANKL serum analysis. RANKL protein levels were measured in serum

obtained by cardiac puncture using commercial ELISA kits (R&D Systems).

Contact hypersensitivity (CHS). CHS experiments were performed

as described27.

RANK-Fc blocking studies. To inhibit RANK-RANKL interaction in vivo,

K14-RANKL Tg and wild-type mice were intravenously injected with 100 mg

RANK-Fc (Sigma Aldrich) once per week for 4 weeks. Control groups received

100 mg rat IgG antibody (BD Pharmingen). CD40L/K14-RANKL double-Tg

mice were treated with 100 mg RANK-Fc once per week for 6 weeks beginning

at 16 weeks of age. In contact hypersensitivity experiments, mice were injected

intravenously with 100 mg RANK-Fc once per week for 2 weeks.

Systemic autoimmunity. Development of autoimmunity was determined

as described12,43,44.

Cell preparations and flow cytometry. Single-cell suspensions of spleens,

lymph nodes and thymi were prepared as described12,27. In some experiments,

wild-type or K14-RANKL Tg mice were injected intravenously with 1 mg

antibody to mouse IL-2 (clone S4-B6, ATCC)45. For harvesting LCs, mouse ears

were split into dorsal and ventral sides, and LC were allowed to migrate out of

the epidermis into culture medium containing CCL21 (50 ng/ml; R&D

Systems) for 3 d. The expression of cell surface and intracellular markers was

analyzed as described43,44 using antibodies to CD205 (clone NLDC145),

CD45RB (clone 16A), CD62L (clone Mel-14), MHC class II (clone M5/114),

CD103 (clone 2E7), PD1 (clone 34), CD25 (clone PC61), CTLA-4 (clone

UC10-4F10-11), CD86 (clone GL1), CD4 (clone RM4-5) and CD11c (clone

HL3) (all from BD Pharmingen); and antibodies to neuropilin-1 (clone H-286;

Santa Cruz Biotechnology), GITR (R&D Systems), Foxp3 (clone FJK-16s,

eBioscience), Va2 TCR (Invitrogen) and CD207 (clone 929F3). Isotype-

matched control antibodies were included in each staining. Apoptotic cells

were identified using an annexin-V apoptosis detection kit (BD-Pharmingen).

Proliferation assays. CD4+CD25– and CD4+CD25+ cells were sorted by MACS

(Miltenyi) and stimulated with 1 mg/ml antibody to CD3 (clone 2c11) and

1 mg/ml antibody to CD28 (clone 37.51; both BD Pharmingen). We added

1 mCi 3H-thymidine per well and measured thymidine incorporation. Recom-

binant mouse IL-2 (15 U/ml; R&D Systems) was added to the assays. In some

proliferation assays, we used a transwell system with 0.3 mm pore size (BD

Falcon) to evaluate contact dependency of suppression. For some proliferation

assays, T cells were incubated with 1mM carboxy-fluorescein diacetate succini-

midyl ester (CFSE) and cocultured with DCs or LCs before flow cytometric

analysis of proliferation. Dead cells were excluded using TOPRO-3 iodide

(Molecular Probes) labeling.

Cytometric bead array (CBA). The cytokine activity in culture supernatants of

CD4+CD25+ cells, CD4+CD25– cells, and LCs from K14-RANKL Tg and wild-

type mice was assayed by CBA (BD Pharmingen).

Mixed lymphocyte reactions. Allogeneic T cells were mixed with DCs or LCs

and proliferation of T cells (H-2d) was assessed by 3H-thymidine incorporation.

Thymectomy and thymus transplantation. Three-day-old mice were anesthe-

tized and thymectomy was performed by aspiration of both thymic lobes

through a small incision in the skin of wild-type or K14-RANKL Tg mice

above the sternum. The incision was closed using histoacryl tissue adhesive

(Aesculap). Sham-thymectomized mice underwent the same procedure, except

that the thymus was left intact. Thymus grafting was performed by placing two

lobes of neonatal thymus under the left kidney capsule of thymectomized mice.

Generation of bone marrow–derived DCs. DCs were generated as described46.

From day 6 to day 9 of culture, cells were stimulated with 100 ng/ml RANKL

(R&D Systems) or 100 ng/ml LPS, or left unstimulated. On day 9, DCs were

pulsed with 2 mg/ml OVA peptide and injected subcutaneously into OT-2Tg

mice at weekly intervals.

Other methods. Generation of K14-RANKL Tg mice, skin transplantation, LC

migration and quantitative real-time PCR are described in Supplementary

Methods online.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSWe thank M. Voskort and B. Geng for technical assistance. The Tnfs11 (Rankl)cDNA was provided by L. Galibert (Immunex), and the antibody to CD207 byS. Saeland (Institut National de la Sante et de la Recherche Medicale (INSERM)Lyon). This work was supported by the German Research Association (SFB 293,BE 1580/6-2 and BE 1580/7-1 to S.B.; KU 1559/1-1 to A.K.), the InterdisciplinaryCenter of Clinical Research (Lo2/017/07 to K.L. and S.B.), and by the InnovativeMedical Research fund of the University of Munster Medical School (Lo11/06 03to K.L. and S.B.). J.M.P. is supported by grants from the Austrian NationalBank, the Austrian Academy of Sciences, the Austrian Ministry of Science andEducation, the 6th EU framework (EuroThymaide), an EU Marie Curie Excellencegrant, and a program project grant from the Austrian Fonds zur Foerderung derWissenschaftlichen Forschung (FWF).

AUTHOR CONTRIBUTIONSK.L. performed cell-culture experiments and animal experiments, generatedfigures, and helped to write the manuscript. A.M. generated the K14-RANKLTg transgenic mouse strains. S.L. bred and provided the Tnfs11–/– mice.J.A. assisted with human immunofluorescence experiments. A.K. assisted withskin samples and provided expertise in immunohistochemistry.S.G. provided methodological expertise and helped design some experiments.T.S. provided expertise in ultraviolet animal experiments. J.M.P. analyzed thedata and helped design the study and write the manuscript. S.B. (principalinvestigator) designed the experiments, interpreted and analyzed data, anddrafted and edited the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Medicine websitefor details).

Published online at http://www.nature.com/naturemedicine

Reprints and permissions information is available online at http://npg.nature.com/

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