of November 23, 2018.This information is current as

T CellsδγReceptor on Epidermal Role for E-Cadherin as an Inhibitory

Takuro KanekuraYouhei Uchida, Kazuhiro Kawai, Atsuko Ibusuki and

ol.1003853http://www.jimmunol.org/content/early/2011/05/11/jimmun

published online 11 May 2011J Immunol 

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The Journal of Immunology

Role for E-Cadherin as an Inhibitory Receptor on Epidermalgd T Cells

Youhei Uchida, Kazuhiro Kawai, Atsuko Ibusuki, and Takuro Kanekura

E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as

epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal gd T cells, known as dendritic epidermal

T cells (DETCs), but they express another receptor for E-cadherin, aE(CD103)b7 integrin, as well. In this study, we analyzed

functional differences between E-cadherin–mediated homophilic binding and heterophilic binding of aEb7 integrin to E-cadherin

in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not aEb7 integrin, was downregulated on

activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated

by aEb7 integrin, and blocking of the binding of aEb7 integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable

binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine

production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented

by coligation of aEb7 integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti–E-cadherin Ab, plate-

bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an

inhibitory receptor on DETCs, whereas aEb7 integrin acts as a costimulatory receptor. Differential expression of E-cadherin and

aEb7 integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin

and aEb7 integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes. The Journal of

Immunology, 2011, 186: 000–000.

E-cadherin is a calcium-dependent homophilic adhesionmolecule that plays vital roles in embryonic developmentand maintenance of polarity and integrity of adult epi-

thelial tissues (1, 2). Its cytoplasmic domain interacts with in-tracellular proteins including catenins, which connect E-cadherinto the actin cytoskeleton (1, 2). In the epidermis, E-cadherinconstitutes the major adhesion molecule in intercellular adherensjunctions between keratinocytes (2). Epidermal Langerhans cellsalso express E-cadherin, and the homophilic binding betweenE-cadherin molecules mediates heterotypic intercellular adhesionof Langerhans cells to keratinocytes in vitro (3). E-cadherin–me-diated adhesion to keratinocytes may contribute to retention ofLangerhans cells in the epidermis in vivo because E-cadherin isdownregulated during maturation of Langerhans cells (3) thatleads to their emigration from the epidermis.Resident murine epidermal gd T cells, known as dendritic epi-

dermal T cells (DETCs), also express E-cadherin (4, 5). DETCsexpress a skin-specific invariant Vg3 TCR (6) and are activated toexert various effector functions including immunoregulation (7,8), tumor surveillance (9), and wound healing (10, 11) throughTCR-mediated recognition of undefined self-Ags induced on

neighboring stressed, damaged, or transformed keratinocytes (12,13). Unlike Langerhans cells, blocking of the homophilicE-cadherin binding does not inhibit adhesion of DETCs tokeratinocytes in vitro (4, 5). This is possibly because DETCsexpress another receptor for E-cadherin, aE(CD103)b7 integrin(14).Besides DETCs, aEb7 integrin is expressed on intestinal and

other intraepithelial lymphocytes (IELs), intestinal lamina propriaT cells (15), some effector CD8+ T cells (16–18), and subsets ofregulatory T cells (19), dendritic cells (20), and mast cells (21).Adhesion of intestinal IELs to epithelial cells in vitro is mediatedby heterophilic binding of aEb7 integrin to epithelial E-cadherin(22–27). Because integrin aE-chain–deficient mice have reducednumbers of resident intestinal IELs (28), contribution of aEb7

integrin to generation or maintenance of intestinal IELs in vivothrough binding to epithelial E-cadherin has been suggested.CD8+ T cells stimulated with TGF-b acquire expression of aEb7

integrin (16–18, 29). Interactions of aEb7 integrin with E-cadherinmediate adhesion of CD8+ T cells to epithelial cells in vitro (18)and are crucial for the lysis of E-cadherin–expressing tumor cellsby CTLs (29). In addition, binding of aEb7 integrin to E-cadherinmay provide a costimulatory signal in intestinal IELs and CD8+

T cells because cross-linking of aEb7 integrin leads to enhancedproliferation in response to suboptimal TCR stimulation (14, 25,30).Because DETCs are also reduced in integrin aE-chain–deficient

mice (31), aEb7 integrin has been implicated in retention ofDETCs in the epidermis. In addition, a recent study showed thataEb7 integrin influences cellular shape and motility of DETCs(32). However, roles of aEb7 integrin in the adhesion of DETCs tokeratinocytes, in the DETC-mediated lysis of keratinocytes, or inthe costimulation of DETC activation have not been elucidated.Furthermore, it remains unknown whether homophilic E-cadherinbinding and heterophilic binding of aEb7 integrin to E-cadherinon keratinocytes have distinct functions in DETCs.

Department of Dermatology, Kagoshima University Graduate School of Medical andDental Sciences, Kagoshima 890-8544, Japan

Received for publication November 29, 2010. Accepted for publication April 13,2011.

Address correspondence and reprint requests to Dr. Kazuhiro Kawai, Department ofDermatology, Kagoshima University Graduate School of Medical and Dental Sciences,8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. E-mail address: kazkawai@m2.kufm.kagoshima-u.ac.jp

The online version of this article contains supplemental material.

Abbreviations used in this article: DETC, dendritic epidermal T cell; IEL, in-traepithelial lymphocyte; KLRG1, killer cell lectin-like receptor G1; MFI, meanfluorescence intensity.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003853

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In this study, we analyzed functional differences between E-cadherin and aEb7 integrin expressed on DETCs, both of whichinteract with E-cadherin on keratinocytes. In this article, we showthat E-cadherin and aEb7 integrin expression on DETCs are dif-ferently regulated depending on the activation state, and that theyprovide opposite signals for DETC activation through binding toE-cadherin on keratinocytes.

Materials and MethodsMice

C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). Femalemice were used at 6–12 wk of age. All procedures have been approved bythe Animal Care Committee of Kagoshima University following the in-stitutional guidelines.

Cell preparation

Epidermal cells were prepared as described previously (33) with a fewmodifications. In brief, the ear skin was treated for 40 min at 37˚C with 1%trypsin (Invitrogen, Carlsbad, CA) in PBS containing 1 mM CaCl2. Theepidermis was separated and collected in IMDM (Invitrogen) supple-mented with 10% FCS (JRH Biosciences, Lenexa, KS) and 0.025% DNaseI (Sigma-Aldrich, St. Louis, MO). Single-cell suspensions were obtainedby mechanical agitation and sequential filtration through 70- and 30-mmnylon meshes.

Short-term DETC lines were generated in each experiment by stimu-lating the epidermal cells (1–2 3 106/ml) for 7 d in 24-well plates coatedwith 10 mg/ml anti-TCR Cd mAb (clone UC7-13D5; BD Biosciences, SanJose, CA). DETCs were harvested and restimulated for 24 h with 1 mg/mlCon A (Sigma-Aldrich), followed by expansion for an additional 14 d inIMDM supplemented with 10% FCS, 50 mM 2-ME, and 10 ng/ml mouserIL-2 (R&D Systems, Minneapolis, MN). The resulting cell population thatwas .95% pure TCR Vg3+ DETCs and had been ’’rested’’ for 14 d afterthe last stimulation was used as resting DETCs. Resting DETCs stimulatedfor 24 h with 10 mg/ml immobilized anti-TCR Cd mAb (UC7-13D5) wereused as activated DETCs. At the time of use, cells were harvested by in-cubation for 3 min with 1 mM EDTA in PBS.

The Pam 212 transformed keratinocyte cell line was grown in IMDMsupplemented with 10% FCS. At the time of use, cells were harvested bypipetting after incubation for 5 min with 0.25% trypsin in PBS containing1 mM CaCl2. The Pam 212 cells cultured in our laboratory expressE-cadherin but not ICAM-1 even after 3-d culture in the presence of 50 ng/ml mouse rIFN-g (R&D Systems) (Supplemental Fig. 1).

Flow cytometry

Cells were resuspended in PBS supplemented with 2% FCS and 0.1%NaN3.After preincubation with anti-CD16/CD32 mAb (2.4G2; BD Biosciences),cells were stained with the following mAbs: FITC-, PE-, or biotin-conjugated anti-TCR Vg3 (clone 536; BD Biosciences or BioLegend,San Diego, CA), PE-conjugated anti-integrin aE-chain (2E7; eBioscience,San Diego, CA), PE-conjugated anti-integrin b7-chain (M293; BD Bio-sciences), biotin-conjugated anti-killer cell lectin-like receptor G1(anti-KLRG1) (2F1; eBioscience), FITC-conjugated anti-integrin aL-chain(M17/4; eBioscience), FITC-conjugated anti-integrin aM-chain (M1/70;BD Biosciences), FITC-conjugated anti-integrin aX-chain (HL3; BDBiosciences), FITC-conjugated anti-integrin b2-chain (H18/2; eBio-science), PE-conjugated anti-integrin a4-chain (R1-2; eBioscience), PE-conjugated anti-integrin b1-chain (HMb1-1; eBioscience), PE-conjugatedanti-integrin aV-chain (RMV-7; eBioscience), PE-conjugated anti-integrinb3-chain (2C9.G3; eBioscience), FITC-conjugated anti-CD62L (MEL-14; eBioscience), PE-conjugated anti-CD25 (7D4; BD Biosciences), andFITC-, PE-, or biotin-conjugated isotype controls (BD Biosciences oreBioscience). Biotin-conjugated mAbs were visualized with Alexa Fluor488-conjugated (Molecular Probes, Eugene, OR), PE-conjugated (South-ern Biotechnology, Birmingham, AL), or PE-Cy5–conjugated (eBio-science) streptavidin. For double staining of TCR Vg3 and E-cadherin,cells were preincubated with normal rabbit Igs (DAKO, Glostrup, Den-mark) and stained with PE-conjugated anti-TCR Vg3 (clone 536) andunlabeled anti–E-cadherin (ECCD-2; Takara Bio, Otsu, Japan) or iso-type control mAbs, followed by FITC-conjugated rabbit anti-rat Igs Ab(DAKO) that was preincubated with unlabeled normal hamster IgG(eBioscience). For staining cells with anti–ICAM-1 or anti-integrin a5-chain mAb, cells were preincubated with normal goat IgG (Santa CruzBiotechnology, Santa Cruz, CA) and stained with unlabeled anti–ICAM-1(3E2; BD Biosciences), anti-integrin a5-chain (HMa5-1; a gift from H.

Yagita, Department of Immunology, Juntendo University School of Med-icine, Tokyo, Japan), or isotype control mAb, followed by PE-conjugatedgoat anti-hamster IgG (H+L) F(ab9)2 Ab (Caltag Laboratory, Burlingame,CA). After gating on forward and side scatters and propidium iodide, vi-able cells were analyzed on an EPICS XL flow cytometer with EXPO32software (Beckman Coulter, Fullerton, CA), and data were analyzed usingFlowJo software (Tree Star, San Carlos, CA).

Wounding and immunofluorescence staining of epidermalsheets

Mice were anesthetized with 2,2,2,-tribromoethanol, and three sets of full-thickness wounds through the panniculus carnosus were created on theshaved back skin using a 3-mm punch tool (10). Mice were caged in-dividually and wounds were left uncovered. The periwound skin and thecontrol skin distant from the wounds were collected 24 h after wounding.

Epidermal sheets were prepared as described previously (34) using 0.5 Mammonium thiocyanate and fixed for 20 min in cold acetone. After pre-incubation for 15 min with 10% normal goat serum (Nichirei, Tokyo,Japan), the sheets were incubated overnight at 4˚C with anti–E-cadherin(ECCD-2 or clone 36; BD Biosciences), anti–b-catenin (clone 14; BDBiosciences), or anti-integrin b7-chain (FIB27; BD Biosciences) mAb.After rinses in PBS, the sheets were incubated overnight at 4˚C with AlexaFluor 488-conjugated goat anti-rat IgG (H+L) (for ECCD-2 and FIB27) orgoat anti-mouse IgG (H+L) F(ab9)2 (for clones 36 and 14) Ab (both fromMolecular Probes). The sheets were then stained sequentially with 10%normal hamster serum (Jackson ImmunoResearch, West Grove, PA) for 15min, biotin-conjugated anti-TCR Vg3 mAb (clone 536) for 1 h at 37˚C,and rhodamine-conjugated streptavidin (Southern Biotechnology) for 1 hat 37˚C. The stained sheets were examined under a laser-scanning confocalmicroscopy (FV500; Olympus, Tokyo, Japan).

Adhesion assay

Resting DETCs were labeled for 15 min at 37˚C with 5 mM CFSE (Mo-lecular Probes) in PBS, quenched for 30 min at 37˚C with IMDM sup-plemented with 10% FCS, and resuspended in IMDM supplemented with1% FCS. CFSE-labeled DETCs were preincubated for 15 min with in-dicated blocking mAbs or isotype control mAbs and seeded at 500 cells/mm2 on the confluent monolayer of Pam 212 keratinocytes precultured inchamber slides (Lab-Tek II; Nunc, Roskilde, Denmark). The followingmAbs were used for blocking: 200 mg/ml anti–E-cadherin (ECCD-1;Takara Bio), 20 mg/ml anti–E-cadherin (ECCD-2), anti-integrin aE-chain(M290; BD Biosciences; and 2E7; eBioscience), b7-chain (FIB27), aL-chain (M17/4; eBioscience), b2-chain (H18/2; eBioscience), b1-chain(HMb1-1; a gift from H. Yagita), and b3-chain (HMb3-1; a gift from H.Yagita). DETCs were allowed to adhere to the monolayers for 1 h at 37˚C,and nonadherent DETCs were removed by gentle washes with prewarmedIMDM containing 1% FCS. The number of adherent DETCs in a 0.17-mm2 area was counted using a calibrated ocular grid under a fluorescencemicroscopy (Optiphot; Nikon, Tokyo, Japan), and the density of adherentDETCs was recorded as the mean cell number per square millimeter of 10random fields. The percentage of adherent DETCs was calculated as(adherent DETCs per mm2/500) 3 100 (%).

Cytotoxicity assay

Resting DETC effector cells were seeded at the indicated E:T ratios on Pam212 target cells (1 3 104) precultured overnight in 96-well plates, andcoincubated for 4 h in IMDM supplemented with 1% FCS. Effector cellswere preincubated for 15 min with 20 mg/ml anti–E-cadherin (ECCD-2),anti-integrin aE-chain (M290), aL-chain (H17/4), b1-chain (HMb1-1), b3-chain (HMb3-1), or isotype control mAb before addition to the target cells,without washing out the mAb. The lactate dehydrogenase activity releasedin the supernatants was quantified by colorimetric assay using the Cyto-toxicity Detection kit (Roche, Mannheim, Germany). Specific lysis wascalculated as ([experimental release 2 effector cell control release 2spontaneous release]/[maximum release 2 spontaneous release]) 3 100(%). The spontaneous release was ,1% of the maximum release.

DETC culture on the E-cadherin–coated slides

Chamber slides were coated overnight at 4˚C with 1 mg/ml recombinantmouse E-cadherin (R&D Systems) in PBS containing 1 mM CaCl2.Resting DETCs were seeded on uncoated or E-cadherin–coated slides andcultured for 1 or 24 h. Adherent cells were fixed for 5 min in cold acetoneand stained sequentially with 10% normal goat serum for 15 min; anti–E-cadherin (clone 36), anti–b-catenin (clone 14), or anti-integrin b7-chain(FIB27) mAb overnight at 4˚C; and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) F(ab9)2 (for clones 36 and 14) or goat anti-rat IgG (H+L)

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(for FIB27) Ab for 1 h at 37˚C. The slides were examined under confocalmicroscopy.

Stimulation assays

For stimulating DETCswith immobilized mAbs, 96-well plates were coatedovernight at 4˚C with indicated concentrations of anti-TCR CdmAb (UC7-13D5), and anti–E-cadherin (ECCD-2), anti-integrin aE-chain (M290), orisotype control mAb. For cytokine production assays, resting DETCs (1 3105) were preincubated for 15 min with anti-CD16/CD32 mAb (2.4G2)and stimulated for 4 h with the immobilized mAbs in the presence ofGolgiPlug (BD Biosciences) containing brefeldin A. Intracellular cytokineproduction was analyzed by flow cytometry as described previously (35)using the Cytofix/Cytoperm kit and PE-conjugated anti–IFN-g (XMG1.2)or anti–TNF-a (MP6-XT22) mAb (all from BD Biosciences). For de-granulation assays, resting DETCs preincubated with anti-CD16/CD32mAb were stimulated for 4 h in the presence of GolgiStop (BD Bio-sciences) containing monensin and Alexa Fluor 488-conjugated anti-CD107a mAb (1D4B; eBioscience). After stimulation, viable cells weredirectly analyzed by flow cytometry.

For stimulating DETCs on the E-cadherin–coated plates, 96-well plateswere coated overnight at 4˚C with 1 mg/ml recombinant E-cadherin in PBScontaining 1 mM CaCl2. Resting DETCs preincubated with anti-CD16/CD32 mAb were seeded on uncoated or E-cadherin–coated plates. Im-mediately after seeding or after culture for 24 h on the plates, DETCs werestimulated for 4 h with 1 mg/ml Con A in the presence of GolgiPlug forcytokine production assays or GolgiStop and Alexa Fluor 488-conjugatedanti-CD107a mAb for degranulation assays.

For stimulating DETCs with fixed keratinocytes, Pam 212 keratinocytes(1 3 105) were cultured overnight in 96-well plates, and the keratinocytemonolayers were fixed for 15 min in methanol. Resting DETCs pre-incubated with anti-CD16/CD32 mAb were stimulated for 24 h with thefixed keratinocytes in the presence of 200 mg/ml anti–E-cadherin (ECCD-1) or isotype control mAb. GolgiPlug or GolgiStop and Alexa Fluor 488-conjugated anti-CD107a mAb were added during the last 4 h.

Statistical analysis

Differences between two groups were evaluated by t test. One-way ANOVAwith post hoc multiple comparisons to a control (Dunnett’s procedure) wasused to determine the significance of differences among three groups.Repeated-measures ANOVA was used to compare the cytotoxicity curves,with different E:T ratios serving as repeated measures. All reported pvalues are two-tailed, with a p value ,0.05 considered significant. Sta-tistical calculations were performed using JMP software (SAS Institute,Cary, NC).

ResultsFreshly isolated DETCs express E-cadherin and aEb7 integrinbut not KLRG1

Consistent with previous observations (4, 5, 14), both E-cadherinand aEb7 integrin were expressed on freshly isolated DETCs (Fig.1). Another receptor for E-cadherin, designated KLRG1, that hasbeen identified as an inhibitory receptor expressed on subsets ofNK cells and ab T cells (36, 37) was not expressed on freshlyisolated DETCs (Fig. 1). Besides aEb7 integrin, low levels ofaLb2 integrin and integrin b1-chain, but not a4-chain, which alsopairs with b7-chain, or aVb3 integrin, were expressed on freshlyisolated DETCs that exhibited the resting effector (CD62Llow

CD25low) phenotype (Fig. 1).

E-cadherin, but not aEb7 integrin, is downregulated onactivation of DETCs during wound healing

DETCs play a critical role in wound healing (10). When skinis wounded, DETCs in the periwound skin are rapidly activatedthrough TCR-mediated recognition of unknown self-Ags inducedon neighboring damaged keratinocytes and become round shaped(10). To determine whether the change in cellular shape of DETCson activation in vivo is associated with alterations of E-cadherin oraEb7 integrin expression, or both, we analyzed expression of thesemolecules on DETCs in the periwound skin by immunofluores-cence staining of epidermal sheets. In contrast with the dendriticmorphology of DETCs in the control skin, activated DETCs in the

periwound skin became round 24 h after wounding (Fig. 2). E-cadherin expression on the rounded DETCs was downregulated asconfirmed using two different mAbs that recognize extracellular(ECCD-2) and intracellular (clone 36) domains of E-cadherin(Fig. 2). Membrane staining of b-catenin was also reduced inthe rounded DETCs (data not shown). Although aEb7 integrin hasa role for cellular shape of DETCs (32) and was reported to bedownregulated on DETC activation (38), we could not find ap-parent downregulation of aEb7 integrin on the rounded DETCs(Fig. 2). These results suggest that E-cadherin, but not aEb7

integrin, is downregulated on activation of DETCs during woundhealing. In this model, however, E-cadherin expression on thekeratinocytes in the periwound skin also appeared to be di-minished (Fig. 2). Therefore, it was unclear whether the down-regulation of E-cadherin on rounded DETCs was directly asso-ciated with DETC activation or indirectly resulted from disruptionof the homophilic E-cadherin binding caused by downregulationof E-cadherin on neighboring damaged keratinocytes.

E-cadherin, but not aEb7 integrin, is downregulated onactivation of DETCs through TCR stimulation in vitro

To determine whether downregulation of E-cadherin is intrinsicproperty of activated DETCs, we analyzed the expression levels ofE-cadherin and aEb7 integrin on short-term DETC lines beforeand after TCR stimulation in vitro. Expression of both E-cadherinand aEb7 integrin, but not KLRG1, was also detected on restingDETC lines (Fig. 3A). Other integrins expressed on short-termDETC lines were similar to those on freshly isolated DETCs,except for the upregulation of aVb3 integrin on resting DETCs(Fig. 3A). Although TGF-b upregulates aEb7 integrin expressionand modulates other integrins on resident intestinal IELs andperipheral T cells (16–18, 22, 25, 29, 39–41), TGF-b treatmentdid not affect expression of aEb7 integrin or other integrins onshort-term DETC lines (data not shown). After TCR stimulation,E-cadherin expression on DETCs was significantly downregulated(Fig. 3A, 3B). Downregulation of E-cadherin expression on acti-vation of DETCs became detectable 10 min after TCR stimulation(data not shown), and the expression levels continued to decreasefor 24 h. By contrast, activated DETCs showed no alterationin aEb7 integrin expression (Fig. 3A, 3B). Therefore, DETCsdownregulate E-cadherin, but not aEb7 integrin, on activation, andthe downregulation of E-cadherin is intrinsic property of activatedDETCs.

Short-term adhesion of DETCs to keratinocytes in vitro isprimarily mediated by aEb7 integrin

To determine the roles of E-cadherin–mediated homophilic bind-ing and aEb7 integrin-mediated heterophilic binding in DETC ad-hesion to keratinocytes, we tested inhibitory effects of a panel ofmAbs including two different mAbs to E-cadherin, which selec-tively block homophilic binding between E-cadherin molecules(ECCD-1) and heterophilic binding of aEb7 integrin to E-cadherin(ECCD-2) (27), on adhesion of DETCs to keratinocyte mono-layers in vitro.As reported previously (4, 5), blocking of the homophilic E-

cadherin binding with anti–E-cadherin mAb ECCD-1 did not in-hibit adhesion of DETCs to keratinocytes (Fig. 4). By contrast,blocking of the heterophilic binding of aEb7 integrin to E-cadherin with anti–E-cadherin mAb ECCD-2, anti-integrin aE-chain mAbs, and anti-integrin b7-chain mAb inhibited DETCadhesion to keratinocytes (Fig. 4). Blocking mAbs to aLb2 in-tegrin had no effect (Fig. 4), and blocking mAbs to integrin b1- orb3-chains, or both, also failed to inhibit DETC adhesion to kera-tinocytes (data not shown). These results suggest that short-term

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adhesion of DETCs to keratinocytes in vitro primarily depends onheterophilic binding of aEb7 integrin to E-cadherin but not onhomophilic binding between E-cadherin molecules. HomophilicE-cadherin binding would not play a compensatory role for theDETC adhesion in this assay because anti–E-cadherin mAbECCD-1 did not have an additional inhibitory effect on anti-integrin aE-chain mAb-mediated inhibition (Fig. 4).

Lysis of keratinocytes by DETCs depends on heterophilicbinding of aEb7 integrin to E-cadherin

DETCs are potent CTLs against cutaneous tumor cells in vitro (42)and play an important role in cutaneous tumor surveillance in vivo(9). Although a crucial role for aEb7 integrin in the killing ofE-cadherin–expressing tumor cells by aEb7 integrin-expressingCD8+ CTLs has been demonstrated (29), the role of aEb7 integ-rin in the lysis of transformed keratinocytes by DETCs has notbeen determined.DETCs could efficiently kill Pam 212 transformed keratinocytes,

and the lysis was blocked with anti–E-cadherin mAb ECCD-2 and

anti-integrin aE-chain mAb (Fig. 5). Anti-integrin aL-, b1-, or b3-chain mAb failed to inhibit the lysis (data not shown). Therefore,the lysis of Pam 212 keratinocytes by DETCs depends on aEb7

integrin binding to E-cadherin. Anti–E-cadherin mAb ECCD-1,which blocks homophilic binding between E-cadherin mole-cules, could not be used in this assay because addition of this mAbdisrupts homotypic intercellular adhesion between Pam 212 ker-atinocytes, leading to target cell death in the absence of DETCeffector cells.

Stable binding of cell-surface E-cadherin on DETCs toplate-bound E-cadherin takes a longer time than binding ofaEb7 integrin to the E-cadherin

The dependence of short-term adhesion of DETCs to keratinocytesin vitro on heterophilic binding of aEb7 integrin to E-cadherin, butnot on homophilic E-cadherin binding, indicates that formationof stable binding between E-cadherin molecules takes a longertime than binding of aEb7 integrin to E-cadherin. To examinethis possibility, we cultured DETCs on recombinant E-cadherin–

FIGURE 1. Freshly isolated DETCs express E-

cadherin and aEb7 integrin but not KLRG1. Freshly

isolated epidermal cells were stained with indicated

mAbs. Quadrant settings were determined by staining

with isotype control mAbs. The percentages of cells

for a given phenotype are shown. Representative pro-

files from five independent experiments are shown.

FIGURE 2. E-cadherin, but not aEb7 integrin, is downregulated on activation of DETCs during wound healing. Full-thickness wound was created on the

back skin. Epidermal sheets were prepared from the control skin distant from the wound and periwound skin 24 h after wounding, and double-stained with

anti-TCRVg3 (red) and either anti–E-cadherin (clone ECCD-2 or 36) or anti-integrin b7-chain (green) mAbs. For each staining, five sheets were examined

in three independent experiments, and representative fields are shown. Immunofluorescence stain, original magnification 31100.

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coated slides instead of the keratinocyte monolayers becauseDETCs kill keratinocytes when coincubated for a prolonged pe-riod. We analyzed localization of E-cadherin, b-catenin, and aEb7

integrin at the interface between adherent DETCs and E-cadherin–coated slide surface using confocal microscopy, rather than di-rectly analyzing DETC adhesion, because DETCs can adhere touncoated slides, as well as E-cadherin–coated slides.Supporting the dominant role for aEb7 integrin in short-term

adhesion of DETCs to keratinocytes, aEb7 integrin, but not E-cadherin or b-catenin, was detected at the bottom surface ofDETCs 1 h after seeding on the E-cadherin–coated and uncoated

slides (Fig. 6). Although DETCs adhered equally well to uncoatedand E-cadherin–coated slides 24 h after seeding, only DETCscultured on the E-cadherin–coated slides extended cellular pro-trusions, which is mediated by interactions of aEb7 integrin withE-cadherin (32). Accumulation of E-cadherin at the bottom sur-face of DETCs was observed only when cultured on the E-cadherin–coated slides for 24 h (Fig. 6). Because b-catenincolocalized with E-cadherin in areas of contact between DETCsand E-cadherin–coated slides only at this time point (Fig. 6),stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin occurred after 24-h, but not 1-h, culture in vitro.

FIGURE 3. E-cadherin, but not aEb7 integrin, is downregulated on activation of DETCs through TCR stimulation in vitro. A, Resting DETCs were

stimulated for 24 h with 10 mg/ml immobilized anti-TCR mAb (activated DETCs). Resting and activated DETCs were stained with indicated mAbs (open

histograms). Shaded histograms indicate cells stained with isotype control mAbs. Representative profiles from three independent experiments are shown. B,

Relative geometric mean fluorescence intensity (MFI) was determined as geometric MFI of indicated mAb/geometric MFI of isotype control mAb. Data are

expressed as the mean and SD of three experiments. Significant change in the relative geometric MFI on activation was evident only for E-cadherin (**p ,0.01).

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Engagement of E-cadherin on DETCs with immobilized mAbinhibits cytokine production and degranulation in response toTCR cross-linking, whereas coligation of aEb7 integrinenhances DETC activation in response to suboptimal TCRstimulation

We next examined functions of E-cadherin and aEb7 integrin inactivation of DETCs. Cross-linking of E-cadherin or aEb7 integrinon DETCs alone with immobilized mAb did not induce IFN-g orTNF-a production or exocytosis of cytotoxic granules, which wasidentified by externalization of the CD107a protein (Fig. 7). In-terestingly, cocross-linking of E-cadherin on DETCs with immo-bilized anti–E-cadherin mAb inhibited cytokine production and

degranulation in response to TCR cross-linking (Fig. 7). Bycontrast, coligation of aEb7 integrin augmented DETC activa-tion in response to stimulation with a suboptimal concentration(0.1 mg/ml), but not an optimal concentration (10 mg/ml), ofimmobilized anti-TCR mAb (Fig. 7). These results suggest that E-cadherin on DETCs acts as an inhibitory receptor, whereas aEb7

integrin acts as a costimulatory receptor.

Engagement of aEb7 integrin on DETCs with plate-boundE-cadherin enhances mitogen-induced cytokine productionand degranulation, whereas stable binding of cell-surfaceE-cadherin on DETCs to plate-bound E-cadherin inhibitstheir activation

To determine whether E-cadherin and aEb7 integrin on DETCsalso provide inhibitory and costimulatory signals when boundto E-cadherin, mitogen-induced cytokine production and de-granulation were analyzed in DETCs immediately after seeding orafter 24-h culture on the E-cadherin–coated plates. DETCs stim-ulated for 4 h with Con A immediately after seeding on theE-cadherin–coated plates showed augmented cytokine productionand degranulation as compared with DETCs stimulated on un-coated plates (Fig. 8A). Because only aEb7 integrin but not E-cadherin on DETCs is engaged with plate-bound E-cadherin atthis time point (Fig. 6), the augmented responses are attributableto the costimulatory signal through aEb7 integrin.In contrast, when DETCs were cultured for 24 h on the plates and

then stimulated with Con A after the formation of stable bindingbetween cell-surface E-cadherin on DETCs and plate-bound E-cadherin (Fig. 6), DETCs precultured on the E-cadherin–coatedplates showed reduced responses as compared with DETCs pre-cultured on uncoated plates (Fig. 8B). Thus, homophilic bindingof E-cadherin on DETCs to plate-bound E-cadherin provides aninhibitory signal for mitogen-induced cytokine production and de-granulation, and the inhibitory signal is dominant over the costi-mulatory signal through aEb7 integrin in this assay condition.

Homophilic binding between E-cadherin molecules on DETCsand keratinocytes provides an inhibitory signal in DETCs

It would be important to determine whether homophilic bindingbetween E-cadherin molecules on DETCs and keratinocytes alsoprovides an inhibitory signal in DETCs. When DETCs are coin-cubated with keratinocytes, however, lysis of keratinocytes byDETCs occurs within 4 h even in the presence of blocking mAbsthat inhibit the binding of aEb7 integrin to E-cadherin (Fig. 5), andthe keratinocyte monolayers were completely destroyed within 18h (data not shown). Because formation of stable binding betweenE-cadherin molecules on DETCs and keratinocytes would takea much longer time, we cultured DETCs on the fixed keratinocytemonolayers, which remained intact after 24-h coincubation (datanot shown). Fixed keratinocytes stimulated cytokine productionand degranulation by DETCs, though less efficiently than viablekeratinocytes, when coincubated for 24 h (Fig. 9 and data notshown). Blocking of the homophilic binding between E-cadherinmolecules with anti–E-cadherin mAb ECCD-1 augmented DETCactivation in response to the fixed keratinocytes (Fig. 9). There-fore, E-cadherin on DETCs also acts as an inhibitory receptorthrough binding to E-cadherin on keratinocytes.

DiscussionWe showed that E-cadherin, but not aEb7 integrin, on activated androunded DETCs in the periwound skin is downregulated in vivo.Because E-cadherin on short-term DETC lines is also downreg-ulated on activation in vitro, E-cadherin expression on DETCsis intrinsically regulated depending on the activation state. It is

FIGURE 4. Short-term adhesion of DETCs to keratinocytes in vitro is

mediated by heterophilic binding of aEb7 integrin to E-cadherin but not

by homophilic E-cadherin binding. CFSE-labeled resting DETCs were

allowed to adhere for 1 h to the monolayers of Pam 212 keratinocytes in

the presence of isotype control mAbs (filled bars) or indicated blocking

mAbs (open bars). Adherent DETCs were counted under fluorescence

microscopy, and data are expressed as mean adhesion and SD (%). Rep-

resentative data from three independent experiments are shown. Significant

inhibition of the adhesion as compared with control was evident with the

anti–E-cadherin mAb ECCD-2 and mAbs to integrin aE- and b7-chains

(*p , 0.05, **p , 0.01, ***p , 0.001). There was no significant differ-

ence between adhesion in the presence of anti-integrin aE-chain mAb

(2E7) alone and anti–E-cadherin (ECCD-1) + anti-integrin aE-chain (2E7)

mAbs (p = 0.766).

FIGURE 5. Blocking of the interactions between aEb7 integrin and E-

cadherin inhibits the killing of keratinocytes by DETCs. Resting DETCs

and Pam 212 keratinocytes were coincubated for 4 h at the indicated E:T

ratios in the presence of isotype control or indicated blocking mAb. Data

are expressed as mean specific lysis and SD (%). Representative data from

three independent experiments are shown. Significant inhibition of the

cytotoxicity as compared with control was evident with anti–E-cadherin

and anti-integrin aE-chain mAbs (*p , 0.05, **p , 0.01).

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possible, however, that the change in cellular shape of DETCs inthe periwound skin is attributed to the downregulation of E-cadherin on keratinocytes rather than that on activated DETCs,because during wound healing, keratinocytes at wound marginsundergo epithelial–mesenchymal transition and downregulate E-cadherin (43) as confirmed in this study. In addition to homophilicE-cadherin binding that controls cellular shape through connectionto the actin cytoskeleton (2), heterophilic binding of aEb7 integrin

to E-cadherin also influences cellular shape (32). Therefore, themorphological change of DETCs during wound healing in vivocould result from disruption of E-cadherin–mediated homophi-lic binding and/or aEb7 integrin-mediated heterophilic bindingcaused by downregulation of E-cadherin on DETCs, keratino-cytes, or both.We also showed that aEb7 integrin expressed on DETCs

mediates short-term adhesion and cytotoxicity to E-cadherin–

FIGURE 6. Stable binding of cell-

surface E-cadherin on DETCs to plate-

bound E-cadherin occurs after 24-h, but

not 1-h, culture in vitro. Resting DETCs

were cultured for 1 or 24 h on uncoated

or E-cadherin–coated slides. Adherent

DETCs were stained with anti–E-cad-

herin mAb specific for the intracellular

domain (clone 36), anti–b-catenin mAb,

or anti-integrin b7-chain mAb and ex-

amined under confocal microscopy. Fo-

cus planes are on the bottom of cells.

Representative fields of three indepen-

dent experiments are shown. Immuno-

fluorescence stain, original magnifica-

tion 31200.

FIGURE 7. Engagement of E-cadherin on DETCs

with immobilized mAb inhibits cytokine production

and degranulation in response to TCR cross-linking,

whereas coligation of aEb7 integrin enhances DETC

activation in response to suboptimal TCR stimulation.

Plates were coated with indicated concentrations of

anti-TCR mAb and isotype control, anti–E-cadherin,

or anti-integrin aE-chain mAb. Resting DETCs were

stimulated for 4 h on the plates, and intracellular IFN-

g and TNF-a production, as well as cell-surface

CD107a expression, were analyzed by flow cytometry.

Data are expressed as mean positive cells and SD (%).

Representative data from three independent experi-

ments are shown. Significant increase or decrease in

the cytokine production or degranulation by cocross-

linking of E-cadherin or aEb7 integrin as compared

with control is denoted with asterisks (*p , 0.05,

**p , 0.01, ***p , 0.001).

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expressing keratinocytes in vitro, and provides a costimulatorysignal for cytokine production and degranulation in response tosuboptimal TCR cross-linking and mitogen stimulation, similar toother intraepithelial T cells (14, 18, 22–27, 29, 30). Therefore,aEb7 integrin on DETCs may play critical roles in vivo for re-tention and effector functions of DETCs in the epidermis throughbinding to E-cadherin on keratinocytes. Although E-cadherin onDETCs is not involved in the short-term adhesion of DETCs tokeratinocytes in vitro, our data suggest that this might be because

of the delayed formation of stable homophilic binding betweenE-cadherin molecules on DETCs and keratinocytes as comparedwith heterophilic binding of aEb7 integrin to E-cadherin. Alter-natively, E-cadherin and aEb7 integrin on DETCs could competefor binding to E-cadherin on keratinocytes, and high-affinity bind-ing of aEb7 integrin to E-cadherin might predominate over low-affinity binding between E-cadherin molecules in the short-termadhesion assay. This possibility is unlikely, however, because wefound that homophilic E-cadherin binding cannot compensate forDETC adhesion to keratinocytes in vitro when interactions ofaEb7 integrin with E-cadherin are blocked. Stable homophilic E-cadherin binding may also contribute to retention of DETCs in theepidermis, but presumably in a different way from aEb7 integrin-mediated rapid adhesion that regulates cell motility (32).We have identified a novel role for E-cadherin as an inhibitory

receptor on DETCs. Although, to our knowledge, this is the firstdemonstration of a role for E-cadherin as an inhibitory receptoron lymphocytes, inhibitory functions of E-cadherin in epithe-lial cell growth have been established. E-cadherin is a tumor-suppressor protein, and its loss of expression or function is asso-ciated with uncontrolled cell growth (1). Conversely, restoration ofE-cadherin into epithelial cancer cells leads to an inhibition ofproliferation. In normal epithelial cells, homophilic E-cadherinbinding directly transduces a growth inhibitory signal (44) andhas been postulated to be responsible for the phenomenon ofcontact inhibition. In addition to the inhibitory role for E-cadherinin epithelial cells, ligation of E-cadherin on Langerhans cells hasbeen shown to inhibit their maturation and chemokine production(45, 46). Besides DETCs, murine intestinal IELs (47, 48) and asubset of mast cells (49) express E-cadherin, as well as aEb7 in-tegrin. Although in humans, E-cadherin expression has not beendetected on intestinal IELs (26) or other lymphocytes, some hu-man T cell neoplasms express N-cadherin (50–54). Whethercadherins have inhibitory functions in these cells remains to bedetermined in future studies.Signal transduction pathways that mediate inhibitory signals

from E-cadherin have remained elusive. E-cadherin is an importantregulator of the Wnt/b-catenin signaling pathway through bindingof b-catenin, which is a key signal transducer in this pathway (1).The Wnt pathway does not appear to be involved in the E-cad-herin–mediated inhibition of DETC activation, however, becausecross-linking of E-cadherin on DETCs with immobilized anti–E-cadherin mAb, which induces an inhibitory signal, does not recruitb-catenin in areas of E-cadherin accumulation (unpublished data).The inhibition would not be mediated through segregation ofTCR, other activating/costimulatory receptors, or intracellularsignaling molecules away from the contact site by homophilic E-cadherin binding, because soluble mitogen-induced DETC acti-vation can be inhibited by engagement of E-cadherin only on thebottom surface of cells with plate-bound E-cadherin. A number ofsignaling pathways other than the Wnt pathway can also be trig-gered by homophilic E-cadherin binding (1). Further studies arenecessary to explore the signaling pathways that mediate the in-hibitory signal through E-cadherin on DETCs.Differential expression of E-cadherin on resting and activated

DETCs and opposite signals provided in DETCs by engagement ofE-cadherin and aEb7 integrin, both of which bind to E-cadherin onkeratinocytes, suggest that activation threshold of DETCs is fine-tuned by E-cadherin expression on DETCs or keratinocytes, orboth, in vivo. DETC activation is regulated by the balance be-tween positive and negative signals provided through variousactivating/costimulatory receptors including TCR, 2B4 (55, 56),NKG2D (9, 33, 57), and junctional adhesion molecule-like protein(JAML) (58), and inhibitory receptors including Ly49E, CD94-

FIGURE 8. Engagement of aEb7 integrin on DETCs with plate-bound

E-cadherin enhances mitogen-induced cytokine production and degran-

ulation, whereas stable binding of cell-surface E-cadherin on DETCs to

plate-bound E-cadherin inhibits their activation. A, Resting DETCs were

seeded on uncoated or E-cadherin–coated plates and immediately in-

cubated for 4 h in the absence or presence of Con A. B, Resting DETCs

were precultured on uncoated or E-cadherin–coated plates for 24 h. Cells

were then incubated for 4 h in the absence or presence of Con A. In-

tracellular IFN-g and TNF-a production, as well as cell-surface CD107a

expression, were analyzed by flow cytometry, and data are expressed as

mean positive cells and SD (%). Representative data from three in-

dependent experiments are shown. Significant differences in the cytokine

production or degranulation between DETCs stimulated on uncoated and

E-cadherin–coated plates are denoted with asterisks (*p , 0.05, **p ,0.01, ***p , 0.001).

FIGURE 9. Homophilic binding between E-cadherin molecules on

DETCs and keratinocytes provides an inhibitory signal in DETCs. Resting

DETCs were incubated for 24 h on uncoated plates or on the fixed mon-

olayers of Pam 212 keratinocytes in the presence of isotype control or anti–

E-cadherin (ECCD-1) mAb. Intracellular IFN-g and TNF-a production, as

well as cell-surface CD107a expression, were analyzed by flow cytometry,

and data are expressed as mean positive cells and SD (%). Representative

data from three independent experiments are shown. Significant augmen-

tation of the cytokine production and degranulation in response to the fixed

keratinocytes was evident with anti–E-cadherin mAb as compared with

control (**p , 0.01, ***p , 0.001).

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NKG2A (59), and CD200R (60). In normal skin, E-cadherin andaEb7 integrin may also be involved in the regulation of activa-tion threshold of DETCs through binding to E-cadherin on kera-tinocytes. Selective downregulation of an inhibitory receptorE-cadherin, but not a costimulatory receptor aEb7 integrin, onactivation of DETCs would augment activating signals and leadto exerting effecter functions. Because E-cadherin expression onkeratinocytes is downregulated during wound healing (43), spon-giotic dermatitis (61, 62), and tumor progression of transformedkeratinocytes (63), these environmental insults and cellular stressmay also alter the activation threshold of DETCs.Notably, both E-cadherin and aEb7 integrin are expressed on

fetal thymic precursors of DETCs, and E-cadherin is also ex-pressed on fetal thymic stromal cells (14, 64). DETC precursorsundergo positive selection through TCR-mediated signals in thefetal thymus to maturate and migrate to the skin (65). Therefore,inhibitory and costimulatory signals provided through E-cadherinand aEb7 integrin might also influence the thymic selection pro-cess of DETC precursors. In this context, analysis of thymic de-velopment of DETC precursors in integrin aE-chain–deficientmice will be needed to determine whether impaired developmentof DETC precursors is involved in the reduction of DETCs in thisstrain (31).In summary, we have demonstrated that E-cadherin, but not

-aEb7 integrin, is downregulated on activation of DETCs. Al-though both E-cadherin and aEb7 integrin may contribute to re-tention of DETCs in the epidermis through binding to E-cadherinexpressed on keratinocytes, their binding kinetics is different.Importantly, we have shown that E-cadherin on DETCs actsas an inhibitory receptor, whereas aEb7 integrin costimulatesDETC activation. Another adhesion molecule, junctional adhesionmolecule-like protein (JAML) expressed on DETCs, has beenidentified to be an important costimulatory receptor for DETCactivation through interactions with a tight junction protein, cox-sackie and adenovirus receptor expressed on keratinocytes (58).Therefore, interactions between adhesion molecules expressed onDETCs and keratinocytes are not only involved in adhesion butalso in regulating DETC activation by acting as costimulatory orinhibitory receptors. Together, these novel findings imply potentialfunctions of other adhesion molecules in regulation of activationthresholds of DETCs, as well as other intraepithelial T cells.

AcknowledgmentsWe thank Tomoko Fukushige, Kanayo Gunshin, Narumi Sagara, and Nobue

Uto for technical assistance, Hideo Yagita for providing mAbs, Michael P.

Schon for helpful information on blocking mAbs, and Ayano Takeuchi for

advice on stimulation assays using fixed keratinocytes.

DisclosuresThe authors have no financial conflicts of interest.

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10 E-CADHERIN AS AN INHIBITORY RECEPTOR ON EPIDERMAL gd T CELLS

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