CCR8 regulates contact hypersensitivity by restricting cutaneous dendritic cell migration to the draining lymph nodes.

International Immunology International Immunology Advance Access published October 25, 2014

CCR8 regulates contact hypersensitivity by restricting cutaneous dendritic cell migration to the draining lymph nodes

Manuscript ID: Manuscript Type:

Complete List of Authors:

INTIMM-14-0147.R1

Original Research

15-Oct-2014

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Date Submitted by the Author:

International Immunology

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Yabe, Rikio; Chiba University, ; Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science, Shimizu, Kenji; Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science, Shimizu, Soichiro; The Institute of Medical Science, The University of Tokyo, Azechi, Satoe; The Institute of Medical Science, The University of Tokyo, Choi, Byung-Il; The Institute of Medical Science, The University of Tokyo, Sudo, Katsuko; The Institute of Medical Science, The University of Tokyo, Kubo, Sachiko; Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science, Nakae, Susumu; The Institute of Medical Science, The University of Tokyo, Ishigame, Harumichi; The Institute of Medical Science, The University of Tokyo, Kakuta, Shigeru; The Institute of Medical Science, The University of Tokyo, Iwakura, Yoichiro; Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science,

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Keywords:

CCR8, contact hypersensitivity, dermal dendritic cells, Langerhans cells, migration

Downloaded from http://intimm.oxfordjournals.org/ at North Dakota State University on October 28, 2014

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CCR8 regulates contact hypersensitivity by restricting cutaneous dendritic cell migration to the draining lymph nodes

Rikio Yabe1,2,3,*, Kenji Shimizu1,2,*, Soichiro Shimizu2, Satoe Azechi2, Byung-Il Choi2, Katsuko Sudo2, Sachiko Kubo1,2, Susumu Nakae2, Harumichi Ishigame2, Shigeru Kakuta2 and Yoichiro Iwakura1,2,3,4

Tokyo University of Science, Noda, Chiba 278-0022, Japan 2

Center for Experimental Medicine and Systems Biology, The Institute of Medical Science,

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The University of Tokyo (IMSUT), Minato-ku, Tokyo 108-8639, Japan 3

Medical Mycology Research Center, Chiba University, Inohana Chuo-ku, Chiba 260-8673,

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Japan

Core Research for Evolutional and Technology (CREST), Japan Science and Technology

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Agency, Kawaguchi, Saitama 332-0012, Japan *

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Both authors equally contributed to this work

Address correspondence to: Yoichiro Iwakura

Center for Animal Disease Models, RIBS, Tokyo University of Science, Noda, Chiba 278-0022, Japan Tel/Fax: +81-4-7121-4104

E-mail: [email protected]

Running title: CCR8 suppresses contact dermatitis

Keyword : CCR8, contact hypersensitivity, dermal dendritic cells, Langerhans cells, migration

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Center for Animal Disease Models, Research Institute for Biomedical Sciences (RIBS),

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Abstract Allergic contact dermatitis (ACD) is one of typical occupational diseases in industrialized countries. Although various cytokines and chemokines are suggested to be involved in the pathogenesis of ACD, the roles of these molecules remain to be elucidated. CC chemokine receptor 8 (CCR8) is one of such molecules, of which expression is up-regulated in inflammatory sites of ACD patients. In this study, we found that Ccr8-/- mice

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murine model of ACD, compared to wild-type mice. T cells from Ccr8-/- mice showed enhanced proliferative recall responses and Th1 and Th17 cell populations were expanded in these mice. However, CHS responses were similar between SCID mice adoptively transferred

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with Ccr8-/- and WT T cells, suggesting that CCR8 in T cells is not responsible for the exacerbation of CHS. Notably, skin-resident dendritic cells (DCs), such as Langerhans cells

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and dermal DCs, and inflammatory DCs were highly accumulated in lymph nodes (LNs) of Ccr8-/- mice after sensitization. Consistent with this, Ccr8-/- antigen presenting cells readily

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migrated from the skin to the draining LNs after sensitization. These observations suggest that

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CCR8 negatively regulates migration of cutaneous DCs from the skin to the draining LNs in CHS by keeping these cells in the skin.

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developed severer contact hypersensitivity (CHS) responses to 2,4-dinitrofluorobenzene, a

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Introduction The prevalence of contact allergy is approximately 20% in North America and Western Europe populations, and sometimes these people develop allergic contact dermatitis (ACD), resulting in the impairment of quality of life (1,2). Contact hypersensitivity (CHS), a mouse model of ACD, is triggered by repetitive contact-exposure of low-molecular weight chemicals termed as haptens to the skin. CHS is constituted of two phases (3-5). (i)

migration of DCs into draining lymph

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resulting in the

nodes (dLNs) in a

CCR7-CCL19/CCL21-dependent manner (6). Then, these haptenized protein-bearing DCs sensitize naïve T cells, resulting in the generation of effector/memory T cells in the dLNs. (ii)

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Elicitation phase: second challenge of the same haptens to sensitized individuals causes the activation of memory T cells, resulting in the elicitation of local inflammation. Thus, the

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migration of cutaneous DCs to dLNs during the sensitization phase is crucial in the development of CHS.

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Langerhans cells (LCs) are a subset of DCs, which are specifically localized in skin

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epidermis. Upon antigenic challenge to skin, LCs acquire antigens(s) and migrate to dLNs, where they present the antigens to naïve T cells (7,8). Dermal dendritic cells (dDCs) are

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another subset of epidermal/dermal-localized DCs (9). These DC subsets are the most important APCs for the immune responses in the skin (9,10). In addition, inflammatory DCs (iDCs), which are likely to be differentiated from peripheral blood-circulating monocytes (11), are also suggested to be involved in this response (12). However, pathological roles of each DC subset in the development of CHS are still controversial (13-18). In the elicitation stage, various types of pro-inflammatory cytokines and chemokines are released from immune cells and keratinocytes (KCs) (19,20). Elevated levels of cytokines and chemokines, such as IFN-γ, IL-4, TNF, IL-10, CCL1 (also known as I-309 and TCA-3), are detected in the skin biopsies from ACD patients sensitized with nickel (21). Notably, CC 3


Sensitization phase: exposure of skin to haptens activates cutaneous dendritic cells (DCs),


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chemokine receptor 8 (CCR8), the chemokine receptor for CCL1 and CCL8, was also up-regulated in the biopsies, suggesting possible involvement of the CCR8-CCL1/CCL8 axis in the pathogenesis of ACD. The chemokine-chemokine receptor system plays important roles in the trafficking of leukocytes from the circulation to specific organs or inflammatory sites (22). CCR8 induces chemotactic migration of cells by activating calcium influx in response to CCL1 (23,24).

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peritoneal adhesions (25), type I diabetes (26) and hepatitis (27) through recruitment of monocytes/macrophages to the inflammatory sites. CCR8 is also expressed in Th2 cells, and mediates chemotaxis and calcium mobilization of these cells (28). Consistent with this, in

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asthma, a Th2-type disorder (29), CCR8+ T cell population is expanded, and Ccr8-/- mice are refractory to experimental allergic airway inflammation (30), suggesting that CCR8 is

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involved in the pathogenesis of Th2-mediated diseases. Recently, Islam et al. reported that CCL8, a newly identified CCR8 agonist, promotes atopic dermatitis in mice by recruiting

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CCR8-expressing Th2 cells (31). On the other hand, CCR8 suppresses septic peritonitis by

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suppressing activation of macrophages (32). A regulatory role of CCR8 is also suggested in ACD, because CCR8-expressing, nickel-specific skin-homing T cells produce IL-10 (21).

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Therefore, the role of CCR8 in the pathogenesis of ACD remains still controversial. In this report, we have generated Ccr8-/- mice, and found that Ccr8-/- mice were more susceptible to 2,4-dinitrofluorobenzene (DNFB)-induced CHS compared with wild-type (WT) mice. This was because migration of DCs from the skin to dLNs was enhanced in Ccr8-/- mice. These results suggest that CCR8 regulates migration of DCs from skin to dLNs in contact allergic inflammation.

Methods 4


CCR8 is expressed in monocytes and macrophages, and is involved in the development of

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Mice Ccr8-/- mice were generated by homologous recombination techniques using E14.1 ES cells as described in Supporting Materials and Methods, and backcrossed to the BALB/cA mice for 8-to-10 generations (Supplementary figure 1). WT and SCID mice were purchased from CLEA Japan. Mice were bred under specific pathogen-free conditions in an environmentally controlled clean room at the IMSUT, and the RIBS. Age- (8-12-week-old)

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institutional animal use committees and were conducted according to the institutional ethical guidelines for animal experiments and safety guidelines for gene manipulation experiments.

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CHS

CHS was induced as described previously (33). Briefly, mice were sensitized by

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painting the shaved abdomen with 50 µl of 0.5% DNFB (Tokyo chemical industry, Tokyo, Japan) in acetone/olive oil (4:1). Five days after first immunization, the mice were challenged

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with 25 µl of 0.2% DNFB on both surfaces of the right ear. Solvent alone was applied to the

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left ear as a control. Twenty-four hrs after second challenge, a small piece of the earlobe was excised using 6-mm punch instrument and weighed. Earlobe swelling was assessed as

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follows: [Increment of earlobe swelling (mg)] = [weight of challenged earlobe (mg)] – [weight of vehicle-treated earlobe (mg)]. Ear thickness was daily measured with a micrometer caliper. Ear swelling was shown as percentage of thickness compared with ears at day 0.

Immunohistochemistry Ear

biopsies

were

fixed

with

10%

neutral

formalin,

dehydrated

paraffin-embedded. Five-µm sections were stained with hematoxylin and eosin (HE).

Antibody titers 5

and


and sex-matched mice were used for all experiments. All experiments were approved by the


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Sera were collected 3 days after second challenge and levels of IgG specific for dinitrophenyl (DNP)-BSA were measured by ELISA-based assay. An aliquot of serum (50 µl) was applied to DNP-BSA-coated 96-well plate, and the plate was incubated at room temperature for 2 hrs. Then, wells were washed and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Zymed, San Francisco, CA) at room temperature for 1 hr, followed by the incubation with substrate phosphatase SIGMA104

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microplate reader (MTP-300, HITACHI, Japan).

Recall proliferation assay

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Single cell suspensions were prepared from dLNs 24 hrs after sensitization with 0.5% DNFB, and were cultured in the presence of indicated concentrations of DNBS (Tokyo

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chemical industry，Tokyo, Japan) in 96-well plates (2 x 105 cells). PMA and ionomycin (50 ng/ml and 500 ng/ml) treated cells were used as the controls. After incubation for 66 hrs, cells

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were labeled with [3H]-thymidine (0.25 µCi/ml, PerkinElmer, Waltham, MA) for 6 hrs, and

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harvested using a Micro 96 cell harvester (Skatron, Sterling, VA). [3H]-radioactivity in acid insoluble fraction was measured using a Micro Beta Systems (Pharmacia Biotech, Piscataway,

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NJ). IFN-γ and IL-17 concentrations in culture supernatants after 72 hr stimulation with DNBS were determined using OptiEIA IFN-γ (BD Pharmingen, San Diego, CA) and Duo set IL-17 (R&D systems, Minneapolis, MN), respectively, according to the manufacture’s protocols. The proportions of Th and Treg cells were determined by flow cytometry as described below.

Isolation of T and B cells by magnetic cell sorter T and B cells were isolated from the spleen and LNs using autoMACS (Miltenyi Biotech, Bergish Gladbach, Germany) with anti-CD90.2- and anti-B220-conjugated 6


(SIGMA-ALDRICH, St. Louis, MO). The absorbance at 415 nm was measured using a

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microbeads (Miltenyi Biotech), respectively, in accordance with the manufacturer’s instructions.

T cell proliferation T cells (2 x 105 cells) were cultured in anti-CD3 (clone 145-2C11; eBioscience, San Diego, CA)-coated-96-well plates. T cell proliferation was determined as described above.

T cells were purified from both spleens and LNs using autoMACS with anti-CD90.2-microbeads (89% pure; 1 x 105 or 1 x 106), and they were intravenously injected

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into recipient SCID mice. After 1 day, these mice were subjected to DNFB-induced CHS. In the case of DNFB-sensitized T cell transfer into WT mice, mice were sensitized by

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painting with 0.5% DNFB. Five days after sensitization, T cells were isolated from dLNs by MACS with anti-CD90.2-microbeads. The resulting cells (2 x 107 cells/mouse; 98% pure)

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were intravenously injected into WT mice. Six hrs after injection, mice were challenged with

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DNFB.

Flow cytometry

Antibodies used for flow cytometric analysis were listed in Table S1. Flow cytometry was performed as described previously (34). Briefly, cells were stained with primary antibodies for 30 min at 4°C after blocking with 2.4G2, followed by secondary antibodies. Cells were analyzed by flow cytometers, FACSCalibur (Becton Dickinson, Sparks, MD) or FACSCantoII (Becton Dickinson) with FlowJo (Tree Star). Dead cells were stained with either 7AAD (SIGMA-ALDRICH) or LIVE/DEAD Fixable Aqua (Invitrogen). For staining of DC subsets, LN cells were prepared by digesting LNs with 200 U/ml type VIII collagenase (SIGMA-ALDRICH). For intracellular cytokine staining, cells were cultured in the presence 7


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Adoptive T cell transfer


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of 50 ng/ml PMA, 500 ng/ml ionomycin and 1 µM monensin for 5 hrs. Cells were washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin, followed by staining with anti-IFN-γ, anti-IL-17 or anti-Foxp3 antibodies. For preparation of highly purified cells, cells were sorted by FACSAria II (Becton Dickinson) or MoFlo XDP (BECKMAN COULTER, Miami, FL).

Tesque,

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RNAs from tissues and cells were purified using the Sepasol-RNA I Super (Nacalai Kyoto,

Japan)

and

GenElute

mammalian

total

RNA

miniprep

kit

(SIGMA-ALDRICH), respectively. Resulting RNAs were reverse-transcribed using the high

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capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). For semi-quantitative analysis, synthesized cDNA was amplified by PCR with Ex Taq DNA

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polymerase (TaKaRa, Kyoto, Japan) and a primer set (Table S2). For quantitative analysis, SYBR Green qPCR kit (TaKaRa) was used with specific primer sets (Table S3).

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Staining of epidermal sheets

Epidermal sheets were obtained from earlobes according to a previous report (35).

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Earlobes were cut off at the base, and split into dorsal and ventral halves. The dorsal sheets were incubated with 20 mM EDTA/PBS for 2 hrs at 37°C. The epidermis were fixed in cold acetone for 20 min, permeabilized in 0.1% saponin/PBS, and blocked with 5% FBS/PBS. After washing, the sheets were stained with 5 µg/ml biotin-conjugated anti-CD207 monoclonal antibody (clone 929F3.01, Dendritics, Dardilly, France), followed by DyLight549-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA). Sheets were mounted with MOUNT-QUICK (DAIDO SANGYO, Saitama, Japan). Images were obtained using a fluorescent microscope, BZ-9000 (KEYENCE, Osaka, Japan). Cell numbers were counted in three independent fields per section of a mouse epidermal sheet. 8


Quantitative reverse-transcription (RT)-PCR

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FITC migration Mice were sensitized by painting with 50 µl of 0.5% FITC isomer I (SIGMA-ALDRICH) in acetone/dibutylphthalate (1:1, vol:vol) on the shaved back skin. After 24 hrs, dLNs were collected and single cell suspension was prepared by digesting with collagenase. Cells were blocked, and were stained with antibodies. The contents of FITC+DC

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Immunofluorescence staining

LNs were embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan) and

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frozen with liquid nitrogen. Seven-µm serial cryosections were fixed with 4% PFA, blocked with 5% goat serum in PBS/0.3% Triton X-100 and stained with hamster anti-mouse CD11c

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antibody (clone, N418; Biolegend, San Diego, CA), followed by DyLight594-conjugated goat anti-hamster IgG (Biolegend). The coverslips were mounted with using ProLong Gold

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Antifade mountant with DAPI (Molecular Probes, Eugene, OR). Images were acquired using

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a fluorescent microscope, BZ-9000.

Isolation of epidermal cells from the skin

Shaved skin was cut out, and subcutaneous adipose tissues were removed by gently rubbing. After washing, the skin was floated on 0.25 mg/ml thermolysin (SIGMA-ALDRICH) for 1 hr at 37°C. Epidermis was peeled off, minced by scissors and disaggregated by vigorous pipetting. Epidermal cells were isolated by a cell sorter.

Generation of bone marrow-derived DCs and macrophages For generation of bone marrow (BM)-derived DCs (BMDCs), BM cells were cultured at 2 × 106 cells/10 ml RPMI1640 supplemented with 10% FBS, 100 U/ml penicillin, 9


subsets were analyzed by flow cytometry.


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100 µg/ml streptomycin, 1% 2-mercaptoethanol and 20 ng/ml recombinant mouse GM-CSF (Peprotech, Rocky Hill, NJ) in 100-mm non-treated dishes. On day 3, 10 ml of the same fresh medium was added to the dishes. On day 6 and 8, 5 ml of the medium was replaced with fresh medium, which contained 10 ng/ml GM-CSF. On day 10, non-adherent cells were collected as BMDCs. For generation of BM-derived macrophages (BMMPs), BM cells were cultured at 5 × 107 cells/10 ml RPMI1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml

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systems) in 100-mm non-treated dishes. On day 3, 5 ml of the same fresh medium was added to the dishes, and on day 7, adherent cells were collected by a cell scraper with 2.5 mM EDTA.

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Statistics

Two-tailed unpaired student’s t-test was used for statistical evaluation of all experiments. P value < 0.05 was considered as significant; * < 0.05; ** < 0.01; *** < 0.001.

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Data were expressed as a mean ± SD.

Results

Ccr8-/- mice are highly susceptible to DNFB-induced CHS

We first investigated Ccr8 mRNA expression in DNFB-induced CHS. Consistent with the observations in ACD patients (21), an increase of Ccr8 transcript was observed in sensitized skin compared with untreated skin (Fig. 1A). To investigate the pathological role of CCR8 in contact dermatitis, we generated Ccr8-/- mice on the BALB/cA background (Supplementary figure 1), and examined the development of CHS. When CHS was induced in the earlobe by challenging DNFB, Ccr8-/- mice developed severer inflammation, as indicated by significantly increased punched earlobe weight at 24 hrs (Fig. 1B). The extent of ear 10


streptomycin, 1% 2-mercaptoethanol and 20 ng/ml recombinant mouse M-CSF (R&D

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thickness in Ccr8-/- mice was significantly increased at 24 hr after immunization (Fig. 1C). Histological analysis showed notable swelling of the earlobe and marked accumulation of granulocytes (Fig. 1D). Analysis of cytokine gene expression revealed that the expression of Il1b gene was significantly enhanced in Ccr8-/- mouse ears compared with WT mouse ears (Fig. 1E), while Tnf and Il10 gene expressions were not. Although IgG antibody levels specific to DNP tended to increase in Ccr8-/- mice compared to WT mice, the difference was

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affect the development of CHS (Fig. 1F) (36).

T cell proliferating response is enhanced in Ccr8-/- mice

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Because Ccr8 gene is highly expressed in T cells (37,38), we examined the proliferating response of Ccr8-/- LN cells. After sensitization of mice with DNFB, dLN cells

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were isolated, and proliferative response to DNBS was evaluated by [3H]-thymidine incorporation. The proliferative response of Ccr8-/- LN cells was significantly higher than that

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of WT LN cells, while PMA/ionomycin-induced or anti-CD3-induced proliferation was

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comparable (Figs. 2A and 2B). Under the experimental conditions, Th1, Tc1 and Th17 cell populations were significantly expanded in Ccr8-/- mouse LNs after sensitization with DNFB

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compared with WT mouse LNs (Fig. 2C). Higher concentrations of IFN-γ and IL-17 were also detected in the culture supernatants from Ccr8-/- LN cell culture (Fig. 2D). The results suggest that T cell priming against DNFB is enhanced in Ccr8-/- mice.

T cells from DNFB-primed Ccr8-/- mice induce enhanced CHS response in WT mice We examined T cell function of Ccr8-/- mice by employing adoptive T cell transfer techniques. T cells were purified from spleens and LNs, and transferred into recipient SCID mice (Fig. 3A, left). After 24 hrs, CHS responses were induced in these mice. We found that earlobe swelling was comparable between WT T cell- and Ccr8-/- T cell-transferred mice (Fig. 11


not statistically significant, consistent with our previous report that antibody levels don’t


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3A, right), indicating that CCR8 deficiency in T cells does not affect the development of contact dermatitis. Next, T cells were purified from dLNs of Ccr8-/- mice 5 days after sensitization, and were intravenously transferred into WT mice. Those mice were then challenged with DNFB (Fig. 3B, left). CHS development was aggravated in Ccr8-/- T cell-transferred mice compared with WT T cell-transferred mice (Fig. 3B, right), indicating that T cell priming is promoted in

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The migration of cutaneous DCs to dLNs is enhanced in Ccr8-/-mice Because adoptive T cell-transfer experiments suggested that cutaneous DCs, but not

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T cells, affect priming responses, we analyzed the DC function in Ccr8-/- mice in CHS. We examined the expression of CCR8 in highly-purified DC subsets, which were separated

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according to Ruedl et al. (39) (purity > 99 %; Supplementary figure 2 for the gating protocol). These DC subsets corresponded to well documented LN DC population (35,39,40). We found

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that Ccr8 mRNA was significantly expressed in DC subsets, including cDCs dDCs

(CD11cintCD40hi),

LCs

(CD11chiCD40hi)

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(CD11chiCD40int),

and

iDCs

(CD11cintCD40int), as well as in T and B cells, but not in bone marrow (BM)-derived LCs

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(BMLCs) and BMDCs (Fig. 4A and Supplementary figure 3).

Because migration of cutaneous DCs into dLNs is a prerequisite to sensitize T cells to induce allergic responses, we analyzed the migration of these DC subpopulations after CHS induction. Significantly enhanced accumulation of dDCs, LCs and iDCs was observed in the dLNs of Ccr8-/- mice compared with WT mice (Figs. 4B and 4C), whereas the proportions of plasmacytoid DCs (pDCs, CD11c+B220+) as well as T and B lymphocytes were similar between WT and Ccr8-/- mice (Supplementary figure 4). In contrast, no significant accumulation of these DC subsets was observed under physiological conditions without allergen challenge (Supplementary figure 5). High expression of MHC-II and CD86, 12


Ccr8-/- mice.

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activation markers for DCs, was similarly observed among WT and Ccr8-/- DC subsets (Figs. 4D and 4E). The expression of CCR7, which is required for the migration of DCs to dLNs through lymphatic vessels (6,35), was also similar in these DCs from WT and Ccr8-/- mice. These results suggest that the migration of cutaneous DCs into dLNs is increased at the sensitization phase of CHS in Ccr8-/- mice. Next, we measured the number of CD207+ DCs in the epidermis using fluorescence

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5A, left). Notably, this reduction was more significantly observed in Ccr8-/- mice (Fig. 5A, right). These observations support the notion that Ccr8-/- DCs migrate more easily from the epidermal compartment to dLNs after sensitization.

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Then, we directly examined migration of cutaneous Ccr8-/- DCs after sensitization with FITC. The proportions of FITC-labeled dDCs, LCs, and iDCs were significantly

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increased in dLNs of Ccr8-/- mice (Figs. 5B and 5C). Immunofluorescent staining analysis revealed that FITC+CD11c+ cells were localized at the cortical area in dLNs, but not at the

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subcapsular sinus (Fig. 6), in consistent with previous reports. (41,42). We also observed that

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the proportion of Ccr8-/- FITC+CD11c+ cells was tended to be higher in that region. These results indicate that CCR8 deficiency promotes migration of DCs from skin to dLNs.

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and

immune

cells.

Epidermal

LCs

(CD45+CD11b+)

and

KCs

(CD45-CD11b-TCRγ/δ-CD117-) were highly purified by flow cytometry (>95% pure), and the expression of Ccl8 was analyzed by quantitative PCR. Ccl8 was expressed in both epidermal 13


microscopy. The number of epidermal DCs was reduced after sensitization with DNFB (Fig.


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LCs and KCs (Fig. 7A). We further compared Ccl8 expression in the skin between steady and inflammatory states. Ccl8 transcript was elevated in DNFB-challenged skin (Fig. 7B), suggesting that CCL8 expression is enhanced during inflammation in the skin.

Discussion

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lymphatic organs (44), while adhesion molecules are important for tethering cells to a tissue (45). In the skin, LCs are anchored to the epidermis through E-cadherin bonds to KCs under steady state conditions (46,47). Upon antigen stimulation, LCs are activated to express CCR7

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and migrate into local dLNs where CCL19 and CCL21 are expressed. CCL8 are also highly expressed in LCs and KCs in the skin. We showed in this report that the expression of CCR8

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on LCs and CCL8 in keratinocytes of the skin was upregulated when the mice were cutaneously sensitized with DNFB, suggesting that the CCR8-CCL8 interaction is important

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for the migration and recruitment of inflammatory cells to the antigen stimulated skin.

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Nonetheless, we showed that Ccr8-/- mice are more susceptible to DNFB-induced CHS, suggesting that CCR8 rather negatively regulates CHS responses. Accumulation of DCs

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including LCs, dDCs and iDCs in the dLNs was significantly enhanced in Ccr8-/- mice compared to WT mice after sensitization. Furthermore, the number of DCs in the skin significantly reduced in DNFB-sensitized Ccr8-/- mice, suggesting that cutaneous Ccr8-/- DCs easily migrated from the sensitized skin to dLNs compared to WT DCs. Consistent with this, we showed that migration of antigen-captured dDCs, LCs and iDCs from the skin to dLN was significantly enhanced in Ccr8-/- mice upon sensitization with FITC. Although CCR8 signaling induces Ca2+ influx and cell activation (23,24,48), we did not find any difference in the expression of the maturation markers, MHC-II and CD86, on DCs between WT and Ccr8-/- mice. These observations suggest that CCR8 expression on APCs is suppressive rather 14


Chemokines mediate the trafficking of leukocytes to inflammatory sites and specific

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than promotive in the development of CHS responses by negatively regulating migration of cutaneous LCs, dDCs and iDCs from skin to dLNs upon stimulation with haptens. It was reported that CCR8 is primarily expressed in Th2 cells (28) and plays an important role in the development of atopic dermatitis by recruiting Th2 cells into allergen-inflamed skin. CCR8 is also expressed in human and murine Treg cells (49-51), which suppress the development of T-cell-mediated skin diseases (52). Therefore, we initially

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inflammatory sites is impaired in the elicitation stage. However, we found that the sensitivity to CHS was comparable between WT T cell-transferred and Ccr8-/- T cell-transferred SCID mice. We also observed that Treg cell population was unchanged in the dLN between WT and

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Ccr8-/- mice after immunization with DNFB (Supplementary figure 6). These results suggest that T cells are not responsible for the exacerbation of CHS in Ccr8-/- mice.

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CCR8 contributes to trafficking of monocyte-derived DCs (MDDCs) from skin to LNs. In an earlier study, Qu et al. (53) showed that the migration of latex-beads+ Ccr8-/-

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MDDCs to subcapsular sinus of the LNs was reduced compared with WT MDDCs. In

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contrast, we found that iDC accumulation in dLNs was significantly enhanced in Ccr8-/- mice at the sensitization phase. This apparent discrepancy could be explained by the difference of

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the models; we examined migration under inflammatory conditions, whereas Qu et al. did not induce inflammation. We found that Ccl8 expression was upregulated in the skin after DNFB-sensitization. Thus, under the physiological conditions, tethering of latex-bead-labeled MDDCs by CCL8 in the skin may not be so strong, and CCL1, which is expressed in the LN subcapsule, is involved in the recruitment of these cells to the dLN. In contrast, when CHS is induced, the migration of cutaneous DCs is strongly restricted by the excess expression of CCL8 in the skin. In conclusion, we have shown here that CCR8 is a negative regulator of APC migration from the skin to dLNs in CHS. This regulation of APC migration may be beneficial 15


speculated that Ccr8-/- mice are highly susceptible to CHS, because Treg cell recruitment to


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for the hosts by keeping APCs in epidermis and preventing excess hapten-induced hypersensitivity (Supplementary figure 7).

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Abbreviations allergic contact dermatitis, ACD; conventional dendritic cells, cDCs; contact hypersensitivity, CHS; dermal dendritic cells, dDCs; draining lymph node, dLN; inflammatory dendritic cells, iDCs; keratinocytes, KCs; Langerhans cells, LCs;

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Funding This work was supported by CREST of the Japan Science and Technology Agency (to Y. I.); the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry (to Y. I.); Grand-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (24220011 to Y. I.); and Grant-in-Aid for JSPS Fellows (11J09956 to R. Y.).

The authors state no conflict of interests.

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Conflict of interest

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References 1

Holness, D. L. 2013. Recent advances in occupational dermatitis. Current opinion in allergy and clinical immunology 13:145.

2

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peripheral blood memory T cells migrating in response to CCL1/I-309. European


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Figure legends Figure 1. DNFB-induced CHS is augmented in Ccr8-/- mice. (A) One day after the second immunization with DNFB or vehicle, the expression levels of Ccr8 mRNA in the skin of a WT mouse was analyzed by semi-quantitative RT-PCR. The data were reproducible in another mouse. (B) Mice were sensitized on their abdomen with DNFB, and 5 days later, mice were challenged with DNFB on the ears. Twenty four hrs later, earlobe swelling was assessed by

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experiment. (C) Ear swelling was quantified by the measurements of ear thickness using a caliper (n = 6-7), and was shown as percentage of thickness compared with ears at day 0. (D) Earlobe sections (5 µm) were stained with HE, and histology was examined by microscopy.

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(E) Cytokine gene expression in the ears from DNFB-immunized mice were analyzed by quantitative PCR (n = 6-7). (F) Serum antibody titers specific for DNP were analyzed by

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ELISA (n = 11-12). The data were reproduced in another independent experiment (F). Means ±SD are shown (B, C, E and F). * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed unpaired

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student’s t-test).

Figure 2. T cell sensitization against DNFB is enhanced in Ccr8-/- mice. (A) Five days after

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sensitization with DNFB, dLN cells were re-stimulated with DNBS. Recall proliferation of LN cells was determined by [3H]-thymidine incorporation (n = 3 each). PMA/ionomycin simulation was used as a control (n = 3 each). The data are representative of three independent experiments. * p < 0.05 (two-tailed unpaired student’s t-test) (B) T cells were isolated from spleen and LNs, and were stimulated with plate-bound anti-CD3 antibody for 3 days. Proliferative response was determined by [3H]-thymidine incorporation. The data are presented as mean ± SD of triplicate wells, and representative of two independent experiments. (C) The proportion of Th1, Tc1 and Th17 cells in dLNs after antigen re-stimulation was analyzed by flow cytometry. CD4+ and CD8+ T cells were stained with 26


the weight of punched earlobes (n = 11-12). The data were reproduced in another independent

Page 27 of 48


anti-IFN-γ or anti-IL-17. Means ±SD of three independent experiments (n = 9 each). * p < 0.05; ** p < 0.01 (two-tailed unpaired student’s t-test). (D) The concentrations of IFN-γ and IL-17 in LN cell culture supernatants were determined by ELISA. Means ±SD of three independent experiments (n = 9 each). ** p < 0.01 (two-tailed unpaired student’s t-test).

Figure 3. T cell sensitization upon treatment with DNFB was enhanced in Ccr8-/- mice

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shown in the left. T cells were isolated from spleens and LNs of Ccr8-/- and WT mice by MACS, and transferred into recipient SCID mice (1 x 105 or 1 x 106 cells/mouse). After 24 hrs, these mice were treated with DNFB to induce CHS. Earlobe swelling was assessed by

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weighing punched earlobes (n = 9-10) (right). The data were reproduced in another independent experiment. (B) Experimental protocol for adoptive transfer of DNFB-sensitized

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T cells (left). T cells were purified from dLNs of DNFB-sensitized mice, and were intravenously transferred into recipient WT mice. After 6 hrs, these mice were challenged

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with DNFB. Increment of earlobe swelling was assessed after 24 hrs (n = 4-5) (right). Means

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±SD. * p < 0.05 (two-tailed unpaired student’s t-test).

Figure 4. Accumulation of dDCs, LCs and iDCs is enhanced in Ccr8-/- dLNs at the sensitization phase. (A) DC subsets were highly purified from dLNs by a cell sorter (99% pure). The gating protocol was described in Supplementary figure 2. The expression of Ccr8 gene in DC subsets was analyzed by real-time PCR. (B) After 24 hr sensitization with DNFB, cells were isolated from dLNs, and DC subsets were analyzed by flow cytometry. The data are shown in (C) (upper panel, percentage; lower panel, number; n = 5 each). The expression of CD86, I-Ad and CCR7 of each population was analyzed by flow cytometry. Representative histogram and the MFI levels of DC markers are shown in (D) and (E), respectively (n = 5

27


compared with WT mice. (A) Experimental protocol for T cell transfer to SCID mice is


Page 28 of 48

each). The data are representative of three independent experiments. Means ±SD are shown (C and E). * p < 0.05; *** p < 0.001 (two-tailed unpaired student’s t-test).

Figure 5. CCR8 regulates migration of cutaneous DCs during the sensitization phase. (A) Epidermal cell sheets were prepared from the earlobe skin after 24 hr-sensitization with DNFB. The sheets were stained with a biotin-conjugated anti-CD207 monoclonal antibody,

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fluorescent microscopy (left; original magnification: x40), and DC numbers are shown in the right (n = 5 each). (B) Mice were sensitized by painting with FITC on the shaved back skin. Twenty-four hrs later, cells were isolated from dLNs, and the contents of FITC+ DCs were

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analyzed by flow cytometry (n = 13 each). The gating protocol was described in Supplementary figure 2. The data are shown in (C) (upper panel, percentage; lower panel,

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number; n = 5 each). The results are reproduced in the two-independent experiments. Means ±SD (A and B). * p < 0.05; *** p < 0.001 (two-tailed unpaired student’s t-test).

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Figure 6. Localization of migratory DCs in dLNs after treatment with FITC. Mice were sensitized with FITC. Frozen sections were prepared from dLNs of these mice, and stained

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with hamster anti-CD11c monoclonal antibody followed by DyLight594-anti-hamster antibody (Red). FITC appears green. Nuclei were stained with DAPI (Blue). Images were acquired by fluorescent microscope. Areas surrounded by dotted lines show the subcapsular sinus (SCS). Original magnification, X20. Left: low-power field, Right: high-power field. Arrows indicate FITC+CD11c+ cells (Yellow).

Figure 7. Ccl8 expression is up-regulated in the skin after sensitization. (A) The expression of Ccl8 gene in cells was analyzed by quantitative PCR. KCs, keratinocytes; LCs, Langerhans cells; MEFs, mouse embryonic fibroblasts; BMDC, bone marrow-derived dendritic cells; 28


followed by DyLight549-streptavidin. Microscopic photographs were taken under a

Page 29 of 48


BMMPs, bone marrow-derived macrophages. (B) WT mice were sensitized with DNFB on the skin. After 1 day, sensitized skin region were isolated. Total RNA was extracted, reverse-transcribed and amplified by quantitative PCR. The data from two independent experiments were combined (n = 10 each). Means ±SD are shown. * p < 0.05 (two-tailed unpaired student’s t-test).

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Supporting Materials and Methods Generation of Ccr8-/- mice The genomic DNA containing CCR8 gene was obtained from a 129/SvJ genomic library (Strategene, La Jolla, CA). The SphI-BglII fragment (1.5 kbp) containing the exon-2, which contains the translation initiation site, was replaced by a neomycin resistance gene (Neo) to disrupt the Ccr8 gene. A diphtheria toxin A fragment under the control of MC1 promoter (DT)

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5’ and 3’ ends are 1.0 and 5.4 kbp, respectively. The targeting vector was linearized by NotI digestion, and electroporated into ES cells (E14.1), and G418 (250 µg/ml; GIBCO, Grand Island, NY) resistant colonies were selected. Homologous recombinants were screened by

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PCR with a forward primer (5’-GGGTTTGAACTGAGGTCTCCATGG-3’) and a reverse primer (5’-TGTGGAATGTGTGCGAGGCCAGAG-3’), and then were confirmed by

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Southern blot hybridization analysis with the 5’ and 3’ probes indicated in Figure S1. An ES clone with correct homologous recombination was used for generation of chimera mice by an

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aggregation method. Germline-transmitted mice were backcrossed to BALB/cA mice for 8-10

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generations. Animal experiments were approved by the institutional committees and conducted according to the institutional ethical guidelines for animal experiments and the

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forward:

Ccr8+/+

reverse:

safety guidelines for gene manipulation experiments. Genotypes were determined by PCR with

the

following

primer-sets;

CCR8

5’-CTGTAATACCTCCACTGAGAAGGC-3’, 5’-CTCACCTTGATGGCATAGACAGCG-3’,

and

common

Ccr8-/-

reverse:

5’-CTACTTCCATTTGTCACGTCCTGC-3’. The lack of Ccr8 transcript was confirmed by quantitative PCR with PCR primers (Table S3).

Generation of bone marrow-derived LCs For generation of BM-derived LCs (BMLCs), BM cells were cultured at 2 × 106 cells/10 ml Iscove's 30


was ligated at the 5’ end of the targeting vector for negative selection. Homologous arms for

Page 31 of 48


Modified Dulbecco's Medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% 2-mercaptoethanol, 25 ng/ml recombinant mouse GM-CSF, 25 ng/ml recombinant mouse IL-4 (Peprotech) and 8 ng/ml recombinant human TGF-β (Peprotech) in 100-mm non-treated dishes. On day 3 and 5, medium were renewed, and on day 7, non-adherent cells were collected as BMLCs.

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Supporting Table 1. Antibodies used in flow cytometric analysis Clone

Manufacturer

FITC-conjugated anti-CD3

145-2C11

Biolegend , San Diego, CA


6D5


PE-conjugated anti-CD11c

N418


APC/Cy7-conjugated anti-B220

RA3-6B2


PE/Cy7-conjugated anti-CD11b

M1/70


APC-conjugated anti-CD40

3/23


APC-conjugated anti-CD4

RM4-5


Biotin-conjugated anti-I-A

AMS-32.1


Biotin-conjugated CCR7

4B12


PerCP-conjugated anti-CD90.2

30-H12


PerCP-conjugated anti-CD19

6D5


AlexaFluor647-conjugated anti-TCRγ/δ GL3


APC/Cy7-conjugagted anti-I-A/I-E


d

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M5/114.15.2

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Pacfic blue-conjugated anti-CD117

2B8


Pacific blue-conjugated anti-B220

RA3-6B2


Brilliant Violet 421-conjugated donkey anti-rabbit polyclonal IgG


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53-6.7

eBioscience, San Diego, CA

PE-conjugated anti-Foxp3

FJK-16s

eBioscience, San Diego, CA


30-F11

BD Pharmingen, San Diego, CA

PE-conjugated anti-CD11b

M1/70

PE-conjugated anti-IFN-γ

XMG1.2

PE-conjugated anti-IL-17

TC11-18H10

Biotin-conjugated anti-CD86

GL1

BD Pharmingen, San Diego, CA BD Pharmingen, San Diego, CA BD Pharmingen, San Diego, CA

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BD Pharmingen, San Diego, CA Invitrogen, Carlsbad, CA

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Pacific blue-conjugated streptavidin

32


Name

Page 33 of 48


Supporting Table 2. Primer sets used in semi-quantitative PCR analysis Reverse

Ccr8

CTCAGAAGAAAGGCTCGCTCAG

CAGCGTGGACAATAGCCAGATACC

Gapdh

ACCACAGTCCATGCCATCTCTGCCA

TCCACCACCCTGTTGCTGTAGCCGT

Ccl8

CGCAGTGCTTCTTTGCCTG

TCTGGCCCAGTCAGCTTCTC

Hprt

AGCTACTGTAATGATCAGTCAACG

AGAGGTCCTTTTCACCAGCA

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Forward


Page 34 of 48

Supporting Table 3. Primer sets used in quantitative PCR analysis

Ccr8

TTCCTCTACTTAGGGAGACAAATGC

CATCCAGGGTGGAAGAATGG

Actb

CAATAGTGATGACCTGGCCGT

AGAGGGAAATCGTGCGTGAC

Ccl8

CGCAGTGCTTCTTTGCCTG

TCTGGCCCAGTCAGCTTCTC

Tnf

GCCTCCCTCTCATCAGTTCT

CACTTGGTGGTTTGCTACGA

Il1b

CAACCAACAAGTGATATTCTCCATG

GATCCACACTCTCCAGCTGCA

Il10

GTGGAGCAGGTGAAGAGTGATTT

TCCCTGGATCAGATTTAGAGAGC

Gapdh

TTCACCACCATGGAGAAGGC

GGCATGGACTGTGGTCATGA

Hprt

AGCTACTGTAATGATCAGTCAACG

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AGAGGTCCTTTTCACCAGCA

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Reverse

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Forward

Page 35 of148 Figure


A

C

DNFB

Ccr8 Gapdh

12

* 10 8 6 4 2 0 WT

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D

Ear swelling (%)

Vehicle

Increment of ear swelling (mg)

B

200 190 180 170 160 150 140 130 120 110 100

Ccr8-/-

***

**

***

WT Ccr8-/0 0

1 24

2 48

Time (h) after 2nd immunization

Vehicle (X 40)

DNFB (X 40)

DNFB (X 100)

rP ev

rR

ee

WT

Ccr8-/-

F

*

1.5 WT WT

1

Ccr8-/Ccr8-/0.5

0 Tnf Tnf

Il1b Il1b

Il10 Il10

Serum anti-DNP-Ab (OD415)

Relative expression (/Hprt)

2

w ie

E

3 72

0.4 0.3 0.2 0.1 0 WT

Ccr8-/-


Figure 2

Page 36 of 48

A 30000

40000

*

*

20000 10000 0

[3H] incorporation (cpm)


Ccr8-/-

30000

20000

10000

0 0

WT

50 DNBS (mg/ml)

Fo

C

25

100

200

2.9

120000 100000 80000

60000 40000 20000 0

PMA/Iono

Ccr8-/-

0 1 Anti-CD3 (mg/ml)

Ccr8-/-

WT 4.2

Ccr8-/-

140000

*

WT

3.0

3.9

0.1

0.1

ee

rP

IFN-g

0.6

1.0

**

6 4 2 0

10000

**

1

0

-/Ccr8 Ccr8-/-

WT WT

D

2

-/Ccr8 Ccr8-/-

WT WT 600

**

**

500 IL-17 (pg/ml)

8000 6000 4000 2000

400 300 200 100

0

0

WT wt

Ccr8 ko-/-

WT wt

CD8 8

IFN-g+CD8+ cells (%)

IL-17+CD4+ cells (%)

IFN-g+CD4+ cells (%)

CD4 8

w ie

ev

rR

IL-17

IFN-g (pg/ml)


WT

B

Ccr8 ko-/-

*

6 4 2 0 WT WT

-/Ccr8 Ccr8-/-



A

Sensitization

Fo

T cells

WT

5

SCID

6

Challenge Measure

SCID

rP

Ccr8-/-

ee

B

WT

T cells WT

2 1 0

1x10^5 1 x 105

Ccr8-/-

1x10^6 10 x 105

24 hr

4

*

3

2

w ie

Ccr8-/-

3

ev

WT

Day 5 6 hr

SCID mice SCID mice

4

rR

Day 0

5


Transfer

0


Day -1

WT T cells Ccr8-/- T cells

1

0

WT WT

Ccr8-/Ccr8-/-


Figure 4

B 7 6 5 4 3 2 1 0

iDCs

0.02 0.01

0.008 0.004

0.4

0.2

1.E+04 104

1.E+03 103 1.E+02 102

1.E+05 105

1.E+02 102

1.E+01 10 Ccr8-/Ccr8-/-

WT WT

LCs

-/Ccr8-/Ccr8

WT

E

iDCs

CD86 (MFI)

CD86

I-Ad (MFI)

I-Ad

Fluorescence Intensity

Ccr8-/-

Isotype control

CCR7 (MFI)

CCR7

1.E+04 104 1.E+03 103 1.E+02 102 1.E+01 10

Ccr8-/Ccr8-/-

dDCs

15000

WT

1.E+02 102

w ie

dDCs

1.E+03 103

1.E+01 10

WT

0.02

1.E+05 105

1.E+04 104

ev

1.E+01 10

*

1.E+03 103

0.04

0

*

rR

LCs (number)

*

0.06

0

ee

1.E+04 104

0.047 (cDC)

0.08

*

0

iDCs (number)

0

***

0.012

rP

LCs (% in LN cells)

* 0.03

0.6 iDCs (% in LN cells)

Fo

dDCs (% in LN cells)

CD11c

0.016

cDCs (% in LN cells)

LCs

0.347 (iDC)

cDCs (number)

dDCs

0.04

dDCs (number)

0.047 (cDC)

0.268 (iDC)

0.008 (LC)

0.02 (dDC)

Ccr8-/-

C

% of Max

Ccr8-/-

0.004 (LC)

0.015 (dDC)

WT

cDCs

D

WT

CD40

Ccr8/Actb

A

Page 38 of 48

WT WT LCs

iDCs

25000

10000

20000

8000

15000

6000

10000

4000

5000

2000

0

0

0

20000

25000

800

15000

20000

600

10000 5000

15000

10000

400

10000

5000

200

5000

0

0

0

3000

2500

400

2000

300

2000

1500

200

1000

1000

100

500 0

WT Ccr8-/-

0

Ccr8-/Ccr8-/-

WT Ccr8-/-

0

WT Ccr8-/-


International Immunology Sensitization

Steady

LCs in epidermal skin (/mm 2)

A WT

Ccr8-/-

900 800 700 600 500 400 300 200 100 0

*

WT WT

Ccr8-/Ccr8-/-

WT WT

Steady

Fo

B

dDC (CD40hiCD11cint)

LC (CD40hiCD11chi)

iDC (CD40intCD11cint)

0.11

0.03

ee

Ccr8-/-

Sensitization

0.05

rP

WT

Ccr8-/Ccr8-/-

CD40

rR

0.09

0.10 0.15

0.1 0.05

0

***

FITC+ iDCs (% of live cells)

0.15

0.2 0.15 0.1 0.05

0

1.E+06 106

-/Ccr8-/Ccr8

WT WT

FITC+ LCs (number)

1.E+05 105 1.E+04 104

1.E+03 103 1.E+02 102 1.E+01 10

WT WT

Ccr8-/Ccr8-/-

1.E+04 104

1.E+03 103 1.E+02 102

WT WT

0.1

0.05

0

1.E+06 106

1.E+05 105

1.E+01 10

***

0.15

WT

**

1.E+06 106

***

0.2

-/Ccr8-/Ccr8

FITC+ iDCs (number)

WT FITC+ dDCs (number)

FITC+ LCs (% of live cells)

0.25

***

w ie

FITC+ dDCs (% of live cells)

0.2

ev

FITC

C

Ccr8-/Ccr8-/-

Ccr8-/Ccr8-/-

**

1.E+05 105 1.E+04 104

1.E+03 103 1.E+02 102 1.E+01 10

WT WT

Ccr8-/Ccr8-/-

WT

Figure 6


SCS

SCS

SCS

SCS

Ccr8-/-

FITC

SCS

SCS

w ie

SCS

ev

rR

DAPI

Merge FITC CD11c DAPI

ee

SCS

rP

Fo

CD11c

Page 40 of 48



B

A

0.6

0.2

4 3 2 1

rR

0

ee

0.4

Ccl8/Hprt

0.8

5

rP

Ccl8/Hprt

1

*

6

Fo

1.2

0 DNFB- Sensitization DNFB+ Steady state state

w ie

ev


A

Page 42 of 48

13.7 kbp

Exon 2

Bgl II

Xba I

Bgl II

Bgl II Sph I

Exon 1

Eco RV

Xba I

12.1 kbp

Bgl II

Fo

PGKNeo

PGKNeo

ee

5’ probe

3’ probe

8.8 kbp

rR

5’ probe

B +/+

+/-

C

10

3’ probe

-/-

12.1 kbp

+/-

-/-

w ie

14.1 kbp

8

ev

+/+

13.7 kbp

8.8 kbp

14.1 kbp

Ccr8/Actb

Mutant allele

Bgl II

Bgl II

Xba I

rP

Exon 1

Xba I

DT

Xba I

Targeting vector

Xba I

WT allele

6 4 2 0 WT WT

KO -/Ccr8

Supplementary figure 1 Generation of Ccr8-/- mice. (A) The strategy for the gene targeting of Ccr8. Exon 2 was replaced by a Neo cassette. Probe positions for Southern blotting hybridization are indicated as arrow boxes. (B) Southern blot analyses of WT (+/+), heterozygous (+/-) and homozygous mutant (-/-) mice. Expected bands of WT and targeted allele were indicated. (C) Detection of Ccr8 mRNA in T cells from WT and Ccr8-/- mice by quantitative PCR with the primers shown in (a) ().

Page 43 of 48


Removal of doublet cells 105

100K 50K

103 102 0

50K 100K 150K 200K 250K

FSC

0

LC

CD11b

cDC

103

0 102

103

105

pDC

0 102

103

B220

105

104

104 103

T cell

B cell

102 0

105

w ie

CD11c

104

104

105

ev

102 0

103

CD11c

rR

iDC 102 0

0 102

FSC

104

103

103

50K 100K 150K 200K 250K

105

104

104

102 0

ee

dDC

105

CD40

104

rP

0 0

CD3/CD19

Dead cell staining

150K

Fo

SSC

200K

105

CD3/CD19

250K

0 102

103

104

B220

Supplementary figure 2 The gating strategy for identification of DC subsets in LNs [Ref 36-38]. B220+CD11c+, pDCs; CD11chiCD40int, cDCs; CD11cintCD40hi, dDCs; CD11chiCD40hi, LCs; and CD11cintCD40int, iDCs.

105


Page 44 of 48

9 8

Fo

6

rP

Ccr8/Actb Ccr8/Actb

7

5 4

-/Ccr8-/Ccr8

2 1 0

BMDCs BMDC

BMLCs BMLC

B cells

rR

ee

3

WT

T cells

cDCs

dDCs

iDCs

LCs

w ie

ev Supplementary figure 3 Expression analysis of Ccr8 mRNA in immune cells by quantitative PCR. Messenger RNAs were extracted from indicated cells and reverse-transcribed. Expression levels of Ccr8 transcript were analyzed by quantitative PCR. (normalized to that of Actb)

Page 45 of 48


WT

0.38 69.7

30 20 10 0

CD11b

pDCs(% in LN cells)

0

B cells (% in LN cells)

CD3/CD19 20

40

0.6 0.4

WT

0.2

Ccr8-/-

0

w ie

ev

T cells (% in LN cells)

40

50

B220

rR

60

31.1

ee

80

0.35

rP

67.7

B220 100

Fo

Ccr8-/-

29.3

Supplementary figure 4 Population of T cells, B cells and pDCs in Ccr8-/- mice is normal at the sensitization phase. After 24 hr sensitization with DNFB, cells were isolated from dLNs from WT and Ccr8-/- mice, and were stained with multiple fluorescence-labeled antibodies. The cell populations were analyzed by flow cytometry. The data are representative of three independent experiments (n = 5 each). Means ± SD are shown.

A

0.391

International Immunology 0.99

WT 62.9

33.2 0.412

0.382

0.146

CD40

CD11b

40

0.5 0.4 0.3 0.1 0

20 10

0 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Ccr8-/-

0.2 0.1

ev

0.2

30

WT

rR

dDCs(% in LN cells)

0 0.6

40

ee

20

50

1.4 1.2 1 0.8 0.6 0.4 0.2 0 0.3

0

iDCs(% in LN cells)

60

pDCs (% in LN cells)

60

0.296

CD11c+

B220

LCs(% in LN cells)

80

0.479

rP

0.8 0.6 0.4 0.2 0

w ie

1.E+08 108 1.E+07 107 1.E+06 106

Cell number

cDCs (% in LN cells) T cells (% in LN cells)

B220

32.4

B cells (% in LN cells)

CD3/CD19

63.9

Fo

C

0.370

0.90

Ccr8-/-

B

0.159 Page 46 of 48

1.E+05 105 1.E+04 104

1.E+03 103 1.E+02 102 1.E+01 10 T cells

B cells

pDCs

iDCs

cDCs

dDCs

LCs

Supplementary figure 5 LN cell population in Ccr8-/- mice is normal under the physiological conditions. LN cells were isolated from axillary, brachial and inguinal LNs from WT and Ccr8-/- mice, and were stained with multiple fluorescence-labeled antibodies. Cells were acquired by flow cytometry. (A) Representative dot plots, (B) percentage and (c) cell number. The data (A and B) from two independent experiments are combined (n = 10 each). (C; n = 5 each)

Page 47 of 48


Ccr8-/-

WT

Foxp3

ee

rP

Treg cells (% of LN cells)

Fo

15 10 5 0

WT

WT

Ccr8-/-

Ccr8-/-

w ie

ev

rR

CD4

20

Supplementary figure 6 Treg cell population in the dLN is normal in Ccr8-/- mice after immunization with DNFB. Five days after sensitization of mice (n = 3, each) with DNFB, dLN cells were collected, and re-stimulated with DNBS. After 3 days, cells were stained for CD4 and Foxp3, and the proportion of Treg cells were determined by flow cytometry. The data are representative of three independent experiments.


Page 48 of 48

Elicitation phase

Sensitization phase

Hapten

Hapten

Skin Inflammatory cytokine chemokine

CCR8

CCR7 Cutaneous DCs

Effector T cells

ee

Migration

rP

Fo

CCL8

CCL19/21

rR

T cells

Memory T cells

w ie

ev

Lymph node

Supplementary figure 7 The role of CCR8 in allergic contact inflammation. Upon sensitization by a hapten, CCL8 expression is upregulated in cutaneous cells such as LCs and KCs. CCR8+ cutaneous APCs are tethered to the skin through CCL8 to prevent excess sensitization of T cells in the dLNs.

Taking the lymphatic route: dendritic cell migration to draining lymph nodes.

Melanoma cell lysate induces CCR7 expression and in vivo migration to draining lymph nodes of therapeutic human dendritic cells.

Leishmania amazonensis infection impairs dendritic cell migration from the inflammatory site to the draining lymph node.

FTY720 Abrogates Collagen-Induced Arthritis by Hindering Dendritic Cell Migration to Local Lymph Nodes.

Dermal tumour necrosis factor-alpha induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans' cell migration.

Antigen-bearing dendritic cells in the draining lymph nodes of contact sensitized mice: cluster formation with lymphocytes.

Immune complexes stimulate CCR7-dependent dendritic cell migration to lymph nodes.

Resolvin E1 inhibits dendritic cell migration in the skin and attenuates contact hypersensitivity responses.

Induction of contact sensitivity. Selective induction of delayed hypersensitivity by the injection of cells from draining lymph nodes into the footpads of normal recipients.

First genomic analysis of dendritic cells from lung and draining lymph nodes in murine asthma.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Guérin.

Lymphocyte migration into lymph nodes.

iodide symporter.

Sarcomatoid tumours of lymph nodes showing follicular dendritic cell differentiation.

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes.

Microvascular changes in lymph nodes draining skin allografts.

Imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Dental pulp dendritic cells migrate to regional lymph nodes.

Sentinel Lymph Nodes in Cutaneous Melanoma.

Cryo-ablation improves anti-tumor immunity through recovering tumor educated dendritic cells in tumor-draining lymph nodes.

TIM-4 is differentially expressed in the distinct subsets of dendritic cells in skin and skin-draining lymph nodes and controls skin Langerhans cell homeostasis.

B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes.

BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming.