Occludin Expression in Epidermal

Occludin Expression in Epidermal δγ T Cells in Response to Epidermal Stress Causes Them To Migrate into Draining Lymph Nodes This information is current as of October 2, 2021. Takahito Saito, Michihiro Yano, Yutaro Ohki, Michio Tomura and Naoko Nakano J Immunol published online 31 May 2017 http://www.jimmunol.org/content/early/2017/05/31/jimmun

ol.1600848 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2017/05/31/jimmunol.160084 Material 8.DCSupplemental http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 31, 2017, doi:10.4049/jimmunol.1600848 The Journal of Immunology

Occludin Expression in Epidermal gd T Cells in Response to Epidermal Stress Causes Them To Migrate into Draining Lymph Nodes

Takahito Saito,* Michihiro Yano,* Yutaro Ohki,* Michio Tomura,† and Naoko Nakano*

Epidermal gd T cells that reside in the front line of the skin play a pivotal role in stress immune surveillance. However, it is not clear whether these cells are involved in further induction of immune responses after they are activated in dysregulated epidermis. In this study, we found that activated gd T cells expressed occludin and migrated into draining lymph nodes in an occludin- dependent manner. Epidermal gd T cells in occludin-deﬁcient mice exhibited impairments in morphology changes and motility, although they expressed activation markers at levels comparable to those in wild-type cells. Occludin deﬁciency weakened the induction of allergen-induced contact hypersensitivity, primarily as the result of the impaired migration of epidermal gd T cells.

Thus, occludin expression by epidermal gd T cells upon activation in response to epidermal stress allows them to move, which Downloaded from could be important for augmentation of immune responses via collaboration with other cells. The Journal of Immunology, 2017, 199: 000–000.

he epidermis provides a barrier against insults from the gdTCRs expressed in DETCs signal constitutively and form the environment, including pathogens, UV light, and chem- immunological synapse at squamous keratinocyte tight junctions

T icals. There are two types of immune cells in murine (16). This semiactivated state may be important for interactions http://www.jimmunol.org/ epidermis: Langerhans cells (LCs), which capture invading pathogens between DETCs and keratinocytes. and bring them to the draining lymph nodes, leading to immune re- In stressed epidermis, DETCs are further activated in response to sponses, and dendritic epidermal gd T cells (DETCs), which recog- molecular stress signals expressed in that layer. Several costimu- nize stress- or damage-induced molecules in keratinocytes (1, 2). latory molecules involved in the interaction of DETCs and stressed DETCs have been proposed to eliminate stressed or DNA-damaged keratinocytes have been identiﬁed. For example, DETCs express keratinocytes, thereby helping to prevent tumor development (3). In junctional adhesion molecule–like protein (JAML), which inter- addition, in wounded epidermis, DETCs secrete cytokines and play acts with Coxsackie and adenovirus receptor (17). In addition, key roles in wound healing, thereby contributing to epidermal ho- DETCs upregulate CD100, which induces costimulatory signals

meostasis (4, 5). Recent work showed that gd T cells promote contact by interacting with plexin B2 on keratinocytes (18). Activated by guest on October 2, 2021 hypersensitivity (CHS) responses (6, 7), skin graft rejection (8), and DETCs undergo morphological changes and detach from tight Ab production (9, 10). However, the molecular mechanisms by which junctions, subsequently reorienting toward basal layers and LCs DETCs activate these immune responses are not well defined. (16). Pathogens and other insults, including chemicals that disrupt DETCs are generated in the first wave of Vg locus rearrange- tight junctions, are also sensed by gdTCRs expressed in DETCs. ments, expressing canonical Vg5Vd1 TCR [nomenclature of Heilig Although there is some evidence that gd T cells function as innate- and Tonegawa (11)], and subsequently migrate to the epidermis. like cells that augment adaptive immune responses, it remains These cells develop in the thymus only during the early embryonic unclear how activated gd T cells interact with other immune cells period and must be selected via mechanisms involving Skint-1, and whether they migrate out of the epidermis. In this study, we which is expressed in thymic epithelial cells and keratinocytes found that DETCs express occludin, a tight junction molecule, in (12–14). Thymocytes expressing this canonical gdTCR engage UVB-irradiated or allergen-treated epidermis. Occludin-deficient Skint-1+ cells and upregulate Egr-3 expression, thus becoming DETCs exhibited impairments in morphological changes and IFN-g–producing gd T cells (15). In the epidermis at steady-state, motility upon activation. Furthermore, allergen-induced CHS responses in occludin-deficient mice were much weaker than those *Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba in wild-type mice. These results suggested that DETCs exert their 278-0022, Japan; and †Department of Pharmaceutical Sciences, Osaka Ohtani Uni- functions by expressing occludin, causing them to become motile versity, Osaka 584-8541, Japan and ultimately leading to the activation of other immune cells. ORCID: 0000-0003-3499-9900 (T.S.). Received for publication May 13, 2016. Accepted for publication May 4, 2017. Materials and Methods This work was supported by a grant from the Center for Technologies against Cancer, Tokyo University of Science. Mice Address correspondence and reprint requests to Dr. Naoko Nakano, Research Insti- C57BL/6J mice were purchased from Sankyo Laboratories (Shizuoka, tute for Biomedical Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Japan). Occludin-deficient mice (Ocln2/2) (19) on the C57BL/6 back- Chiba 278-0022, Japan. E-mail address: [email protected] ground were provided by M. Furuse and were obtained from the Center for The online version of this article contains supplemental material. Animal Resources and Development at Kumamoto University. Littermates +/2 3 +/2 Abbreviations used in this article: CHS, contact hypersensitivity; DETC, dendritic epi- generated from Ocln Ocln crosses were used as occludin-deficient 2/2 +/+ d2/2 dermal gd T cell; DNFB, 2,4-dinitrofluorobenzene; IEL, intraepithelial lymphocyte; (Ocln ) or wild-type control (Ocln ) mice. TCR mice (20) were JAML, junctional adhesion molecule–like protein; LC, Langerhans cell. housed in our mouse facilities. KikGR mice were described previously (21). Six- to ten-week-old male and female mice were used in all exper- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 iments. All mice were maintained under specific pathogen–free conditions in

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600848 2 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION the animal facility at Tokyo University of Science, and experimental studies was exposed to violet light (420 nm) for 2 min using an LED hand lamp were approved by the university’s Animal Care and Use Committee. (LED-420P; OptoCode, Tokyo, Japan). Abs and reagents Real-time RT-PCR Abs against TCRgd (UC7-13D5; BioLegend), CD25 (PC61.5; eBioscience), Total RNA was isolated from cells using ISOGEN and reverse transcribed using CD69 (H1.2F3; eBioscience), CD11c (N418; BioLegend), I-A/I-E (M5/ ReverScript III (both from Wako Pure Chemical Industries). PCR was per- 114.15.2; eBioscience), CD100 (BMA12; eBioscience), CD3 (145-2C11; formed in triplicate using GoTaq qPCR Master Mix containing SYBR Green on eBioscience), or Vg5 (7-17; BD Pharmingen), conjugated to FITC, PE, an Applied Biosystems 7500 Fast Real-Time PCR System. Experiments were allophycocyanin, or biotin, were obtained from the indicated suppliers. independently repeated two or three times. Values for each gene were nor- Allophycocyanin-, PE-Cy7–, or Brilliant Violet 421–conjugated streptavidin malized to the corresponding expression levels of b-actin. The specific primers (BioLegend) was used for the second step. CD16/32 (2.4G2) Abs were used used were as follows: claudin-1: 59-ACTCCTTGCTGAATCGAACAGT-39, for Fc blocking. Purified anti-TCRgd (UC7-13D5, functional grade) and 59-GGACACAAAGATTGCGATCAG-39;claudin-3:59-CTCATCGTGGTG- anti-CD3 (145-2C11, functional grade) were purchased from eBioscience. TCCATCC-39,59-ATGGTGATCTTGGCCTTGG-39; claudin-4: 59-GGAA- Rabbit anti-occludin (711500; Invitrogen) and Alexa Fluor 647– or Alexa TCTCCTTGGCAGTCCT-39,59-CACCCACGATGATGCTGAT-39; claudin-6: Fluor 488–conjugated goat anti-rabbit IgG (A21245; Invitrogen) were used 59-TGTGTGGTTCAGAGCACTGG-39,59-AGCAGACAGGAATGAGCGTC-39; for histochemistry. 2,4-Dinitrofluorobenzene (DNFB) was purchased from claudin-7: 59-ACGCCCATGAACGTTAAGTACGAG-39,59-CTTTGCTTTC- Sigma-Aldrich. PMA (50 ng/ml) and ionomycin (0.5 mM; both from Sigma- ACTGCCTGGACA-39; claudin-8: 59-TCAGAATGCAGTGCAAGGTC-39,59- Aldrich) were used to stimulate cells. AGCCGGTGATGAAGAAGATG-39; ZO-1: 59-AGCGATTCAGCAGCA- ACA-39,59-GGCTCAGAGGACCGTGTAAT-39; ZO-2: 59-GGAAGATG- Cell culture TGCTCCATTCG-39,59-TGCCGACTCCTCTCACTGTA-39; cadherin 1: Epidermal gd T cell lines were established from wild-type, Ocln2/2, 59-CTGCCACCAGATGATGATACC-39,59-CGAACACCAACAGAGAG- KikGR, and KikGR/Ocln2/2 mice by culturing epidermal cells in RPMI TCGTA-39; occludin: 59-ATGACATGTATGGCGGAGAG-39,59-ATA-

1640 complete medium containing 5% FCS and 10 ng/ml IL-2. Epidermal GCCTCTGTCCCAAGCAA-39;tricellulin:59-GTACTCGTGGGGCTGG- Downloaded from stromal cells were obtained from a long-term epidermal cell culture that ATT-39,59-GAACATCGCATTCATTGGTG-39;JAML:59-GTGGTCC- contained heterogeneous cell populations and maintained in RPMI 1640 AGGACGAATTTCA-39,59-ATGCCCTGCTGACCCTTAT-39; b-actin: 59- complete medium containing 5% FCS. GGCTGTATTCCCCTCCATCG-39,59-CCAGTTGGTAACAATGCCATGT-39; HPRT: 59-GTAATGATCAGTCAACGGGGGAC-39,59-CCAGCAAGCTTG- UVB irradiation and tape stripping CAACCTTAACCA-39;andIFN-g:59-ATGAACGCTACACACTGCATCTT- 39,59-GAATCAGCAGCGACTCCTTTTCC-39. To obtain UVB-irradiated epidermis, euthanized mice were placed in a GS

Gene Linker UV Chamber (Bio-Rad), and their ear skin was irradiated at Chemotaxis assay http://www.jimmunol.org/ 2 130 mJ/cm on the dorsal and ventral sides. In cell-migration experiments, + 2/2 dorsal back skin of anesthetized mice was clipped and irradiated at 130 mJ/ Vg5 cells were isolated from wild-type and Ocln DETC lines by cm2. Epidermal mechanical stress was administered by stripping the epi- FACS. Cells were then suspended in 100 ml of culture medium containing 3 6 dermis five times with normal Scotch tape. IL-2 at 1 10 per milliliter and placed into the upper chamber of a 24-well Transwell plate with a 5-mm-pore filter (Corning Costar). Cells Immunohistochemistry that migrated into the lower chamber, which contained 600 ml of culture medium with or without 500 ng/ml CXCL12 (BioLegend), were harvested Dorsal and ventral sides of ear skin were incubated for 1 h at 37˚C in 0.2% after 4 h and counted. Migration of cells from the skin was analyzed by EDTA/PBS, and epidermal sheets were peeled off. These sheets were placed isolating ear skin from wild-type and Ocln2/2 mice. In six-well plates, in slide glasses, fixed in cold acetone for 10 min, rinsed in PBS, and blocked dorsal and ventral sides of skin were floated dermal side down on 4 ml of in 5% BSA/PBS. Samples stained with Abs against Vg5 were analyzed by medium containing 100 ng/ml CCL21 (R&D Systems). Dendritic cells and by guest on October 2, 2021 fluorescence microscopy (KEYENCE BZ-9000). For staining of occludin in gd T cells migrating out of the dermis and epidermis were collected after epidermal sheets, ventral sides of skin were fixed in cold ethanol for 30 min, 48 h and counted and analyzed by FACS. rinsed in PBS, and incubated in 3.8% ammonium thiocyanate/phosphate buffer for 17 min at 37˚C. These samples were rinsed in PBS, and epider- Statistics mal sheets were peeled off. Samples were treated for 1 h with blocking , buffer containing 5% goat serum, 10% FCS, 0.3% Triton X-100, and Fc- Statistical analyses were performed using the Student t test. A p value 0.05 block in PBS and then stained overnight at 4˚C with primary Abs. Following was considered statistically significant. staining with secondary Abs for 1 h at room temperature, the samples were analyzed using an Olympus FluoView FV10i confocal microscope. Results Flow cytometry DETCs are activated in dysregulated epidermis Epidermal cell suspensions were prepared from epidermal sheets isolated by UVB is an environmental insult that affects epidermal homeostasis. incubating ear skin for 1 h at 37˚C in 0.2% trypsin in PBS. Cells prepared To investigate the roles of DETCs in stress surveillance in the from lymph nodes or epidermis were incubated with anti-CD16/CD32 to epidermis, we irradiated C57BL/6 mice with UVB and analyzed block Fc binding and then stained with anti-TCR gd (GL3; BioLegend), activation of DETCs. Epidermal sheets from ear skin were isolated, anti-Vg5 (7-17; BD Pharmingen), anti-CD11c (N418; BioLegend), anti–I- A/I-E (M5/114.15.2; eBioscience), anti-CD100 (BMA12; eBioscience), and and DETCs were stained with FITC-conjugated anti-Vg5 Abs. anti-CD69 (H1.2F3; eBioscience) in various combinations. Cells were DETCs, which were distributed evenly at steady-state, became subjected to flow cytometry on a BD FACSCalibur or BD FACSCanto II, less frequent and lost their dendrites 48 h after irradiation and the resultant data were analyzed with CellQuest Pro or FlowJo software. (Fig. 1A). These cells extended their dendrites toward the surface Contact hypersensitivity of the skin at steady-state, whereas they became rounded in UVB- irradiated epidermis. These morphological changes were observed To analyze CHS responses, we followed the protocol of Nielsen et al. (7), previously in wounded epidermis (22) and in epidermis over- with minor changes. Briefly, 25 ml of 0.15% DNFB in a 1:4 olive oil/ acetone mixture was painted on the dorsal side of one ear, and olive oil/ expressing an NKG2D ligand (2). DETCs sorted from UVB- acetone mixture (vehicle) alone was painted on the other ear, for three irradiated epidermis expressed high levels of IFN-g, indicating consecutive days. On day 23, the ears were painted with the same mate- that they were activated by an unidentified molecule expressed in rials, and 24 h later the ear thickness of anesthetized mice was measured on this tissue following insult (Fig. 1B). both sides using an engineer’s micrometer. Data were expressed as the ear swelling response above baseline (i.e., the thickness of the DNFB-painted Occludin is expressed in activated DETCs ear 2 the thickness of the vehicle-painted ear). DETCs survey stress-induced molecules by extending their den- Photoconversion of skin cells drites into tight junctions and receive signals through their TCRs KikGR mice were anesthetized, and hair was removed from the back skin (16). To identify molecules expressed in DETCs that could be (3 cm 3 3 cm) using a hair clipper (THRIVE model 515R). Naked skin involved in recognition or activation, we stimulated a DETC line The Journal of Immunology 3

FIGURE 1. Epidermal gd T cells express occludin upon activation. (A) C57BL/6 mice were anesthetized and irradiated with UVB (130 mJ/cm2). Epidermal sheets prepared 48 h after UVB irradiation or mock treatment were stained with FITC–anti-Vg5 Abs. (B) DETCs were sorted from epidermal cells obtained from ear skin 24 h after UVB irradiation or without such Downloaded from treatment. Relative expression of IFN-g, normalized against b-actin levels, was analyzed by real-time PCR. (C) Expression of tight junction molecules was analyzed. RT-PCR was performed on total RNA extracted from DETCs cultured with PMA plus ionomycin for 24 h (middle lane) or http://www.jimmunol.org/ from unstimulated DETCs (left panel). Epider- mal cells were used as controls (right panel). (D) DETCs were stimulated on plates coated with anti-CD3 Abs (black bars) or PMA plus ionomycin (gray bars). Relative expression of occludin and other representative molecules, normalized against b-actin levels, was analyzed by real-time PCR. (E) Ear skin was isolated from wild-type (WT) and Ocln2/2 mice, with or without UVB irradiation.

Epidermal sheets were peeled off and stained by guest on October 2, 2021 overnight with anti-occludin and allophycocyanin-conjugated anti-TCRgd Abs, and Alexa Fluor 488–conjugated anti-rabbit IgG was used for the second step. One representative image from four independent experiments is shown. Scale bars, 100 mm. DETCs expressing both occludin (green) and TCRgd (red) are indicated by arrows.

established from epidermal cells with PMA plus ionomycin for anti-CD3 Abs or with PMA plus ionomycin and then measured 24 h and then analyzed the expression of tight junction molecules relative expression levels of tight junction molecules by real-time by RT-PCR. Claudin-1, claudin-3, claudin-4, and claudin-8 were PCR. Occludin expression was also induced by TCR engagement expressed in epidermal cells but not in DETCs. Interestingly, (Fig. 1D), suggesting that DETCs express occludin in response to occludin was expressed in DETCs only after activation, whereas Ags. Expression of JAML, an adhesion molecule that interacts ZO-1, ZO-2, cadherin-1, and tricellulin were constitutively ex- with Coxsackie and adenovirus receptor in tight junctions (17), pressed in resting and activated DETCs (Fig. 1C). To determine was upregulated to some extent; however, induction of occludin whether occludin expression was also induced in DETCs receiving was much higher. To determine whether occludin was also signals through the TCR, we stimulated cells on plates coated with expressed in activated DETCs in stress-induced epidermis, we 4 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION stained epidermal sheets isolated from mouse ear skin, exposed or green to red fluorescence, we clipped the back skin of KikGR not exposed to UVB irradiation, with anti-TCRgd and anti-occludin mice and exposed it to violet light for 2 min. Epidermal stress was Abs. Epidermis obtained from Ocln2/2 mice was also used as a induced by UVB irradiation or tape stripping, both of which negative control for occludin staining. Occludin expression was caused activation of DETCs (tape stripping data not shown). detected in some cells in UVB-irradiated epidermis; these occludin+ Lymph node cells were collected from inguinal lymph nodes cells also expressed TCRgd. Occludin was not detected in TCRgd+ 2–5 d after treatment (Fig. 3A). Almost all DETCs in the epi- cells from epidermis not exposed to UVB irradiation. The occludin dermis were photoconverted to red (KikGR-Red) by violet light staining was specific, because no staining was observed in Ocln2/2 exposure (data not shown). In skin-draining inguinal lymph nodes, epidermis. These results suggested that occludin expression was photoconverted cells were detected in 2 d. Of the photoconverted induced in DETCs upon activation and that occludin expressed in KikGR-Red cells present in inguinal lymph nodes, ∼30% were DETCs may have distinct functions. TCRgd+ in mice treated with UVB irradiation or tape stripping. In contrast, mice with no epidermal stress other than violet light Occludin-deficient DETCs exhibit impairments in exposure had fewer KikGR-Red+ gd T cells in draining lymph morphological changes nodes in the skin (Fig. 3B). These results suggested that gd T cells Ocln2/2 mice have little functional defect in tight junctions (19). are stimulated to migrate in response to stress-induced molecules When we analyzed immune cells in the thymus and lymph node, expressed following UVB irradiation or tape stripping. Cells mi- we observed no difference in immune cell populations between grating from the skin to inguinal lymph nodes were counted on Ocln2/2 and wild-type mice (Supplemental Fig. 1). We prepared days 2–5 after the photoconversion. The number of KikGR-Red epidermal cell suspensions from wild-type and Ocln2/2 mice and cells in inguinal lymph nodes peaked on day 4 after photo- analyzed DETCs (TCRgd+ and Vg5+) and LCs (I-A+) by FACS. conversion. More KikGR-Red cells were found in mice subjected Downloaded from There was no difference between wild-type and Ocln2/2 epider- to tape stripping, suggesting that this treatment more efficiently mis for either population (Fig. 2A). Next, we estimated the fre- promoted cell migration from the skin to the draining lymph nodes quency of DETCs in epidermal sheets by counting cells in 100 3 (Fig. 3C). The number of gd T cells migrating from the skin was 100-mm areas. There was no significant difference between wild- also calculated by FACS analysis. The highest numbers of KikGR- type and Ocln2/2 epidermis with regard to the average number of Red+ gd T cells in draining lymph nodes were found on day 4 in DETCs (Fig. 2B). To determine whether Ocln2/2 DETCs were mice subjected to tape stripping (Fig. 3D). These results demon- http://www.jimmunol.org/ activated in response to Ags, we stimulated DETCs from wild-type strated that gd T cells traffic from the skin to the draining lymph and Ocln2/2 mice on plates coated with anti-TCR gd Abs. The nodes in response to epidermal stress. We then stained with anti- activation markers CD25 and CD100 were upregulated equally in Vg5 Abs to determine whether DETCs were present among the wild-type and Ocln2/2 mice (Fig. 2C). Epidermal sheets were migrated gd T cells in the draining lymph nodes. Although cell prepared from UVB-irradiated mice, and DETCs were stained with numbers were small, we could detect TCRgd+Vg5+ cells among anti-Vg5 Abs. Morphological changes were evaluated by deter- KikGR-Red cells in the inguinal lymph nodes on days 4 after tape mining the proportions of cells with more than two dendrites, one or stripping (Fig. 3E). These TCRgd+Vg5+ cells were only detected two dendrites, or no dendrites. After irradiation, DETCs in Ocln2/2 on days 4 and 5 (Fig. 3F). by guest on October 2, 2021 mice had more dendrites than did those in wild-type mice (Fig. 2D). Next, using Ocln2/2 KikGR mice, we investigated whether These results suggested that occludin expressed in DETCs after occludin expressed on activated DETCs was involved in their activation is involved in cytoskeletal rearrangement. migration. Epidermal cells in wild-type and Ocln2/2 mice were To determine whether occludin expressed in DETCs, but not in photoconverted, and the cells that migrated after tape stripping keratinocytes, was responsible for these effects, we induced were analyzed on day 4. The frequencies of photoconverted cells morphological changes in wild-type and Ocln2/2 DETCs in vitro. in draining lymph nodes were similar (Fig. 4A). The number of On wild-type epidermal stromal cells, DETCs from KikGR and Vg5+ KikGR-Red cells in inguinal lymph nodes varied depending KikGR/Ocln2/2 DETC lines were cultured in the presence of on the mice used; however, we could always detect Vg5+ cells in IL-2. DETCs expressing GFP became adherent and visible under wild-type mice on day 4. Few KikGR-Red cells expressing fluorescence microscopy. The DETCs were then incubated with TCRgd and Vg5 were found in inguinal draining lymph nodes in biotin–anti-CD3 Abs, followed by allophycocyanin-streptavidin, Ocln2/2 mice (Fig. 4A, 4B). These results indicated that the major and their morphologies were observed after 4 h. DETCs from defect in Ocln2/2 mice was impaired migration of DETCs. wild-type and Ocln2/2 mice remained attached to the stromal Because occludin is expressed in activated DETCs and in other cells in the culture, whereas DETCs stimulated with anti-CD3 surrounding cells, such as keratinocytes, we then analyzed whether became rounded (Fig. 2E, indicated by arrows). Numbers of the defect in DETC migration in Ocln2/2 mice was due to the lack cells with rounded versus stretched shapes were counted in 10 of occludin expression in DETCs but not in other cells. For this areas (Supplemental Table I), and average percentages were cal- purpose, we tested wild-type and Ocln2/2 DETCs for their ability culated. More than 60% of wild-type DETCs became rounded, to migrate toward CXCL12. CXCL12 promoted the migration of versus ∼25% of Ocln2/2 DETCs, upon stimulation (Fig. 2F). wild-type and Ocln2/2 DETCs; however, the number of migrated These results indicated that occludin expressed on DETCs plays cells was significantly smaller in Ocln2/2 DETCs than in the wild- a role in the morphological changes that take place in these cells. type (Fig. 4C). These results indicated that occludin expressed in DETCs is involved in migration. Occludin is involved in DETC migration Recent analysis by in vivo microscopy demonstrated that occludin Occludin-deficient mice are less sensitive to CHS expressed in gd intraepithelial lymphocytes (IELs) contributes to The impaired traffic of gd T cells in Ocln2/2 mice may affect their migration (23). Our detection of occludin expression in ac- immune responses in the skin. Because the importance of DETCs tivated DETCs prompted us to investigate whether DETCs are in CHS pathogenesis has been demonstrated (7), CHS was an also able to migrate upon activation. To this end, we analyzed appropriate system for investigating whether occludin expressed migration of DETCs from the epidermis to draining lymph nodes in activated DETCs plays a role in immune responses. First, by tracking cells that express KikGR (21). To convert KikGR from we investigated whether occludin was expressed in DETCs in The Journal of Immunology 5

FIGURE 2. Occludin-deficient DETCs exhibit impairments in morphological changes. (A) Epidermal cells were prepared from wild-type (WT) and Ocln2/2 mice and then stained with anti-TCRgd, anti–I-A, and anti-Vg5 Abs. Cells were gated by forward versus side scatter, and these size-gated cells were analyzed for TCRgd, I-A, and Vg5 expression. Representative data from three experiments are shown. (B) Epidermal sheets from WT and Ocln2/2 mice were prepared and stained with anti-Vg5 Abs. Vg5+ cells (DETCs) were counted in 100 3 100-mm areas, and the average numbers from 30 squares are shown. Representative data from three independent experiments are shown. (C) Epidermal cells from WT and Ocln2/2 mice were gd stimulated for 24 h on plates coated with anti-TCR Downloaded from Abs. Cells were first gated by forward versus side scatter, and Vg5+ cells in these size-gated cells were analyzed for expression of CD100 and CD25. Black line, stimulated DETCs; gray line, unstimulated WT DETCs. Representative data from two experiments are shown. (D) Epidermal sheets were prepared from WT 2/2 and Ocln mice 48 h after UVB irradiation. DETCs http://www.jimmunol.org/ were stained with FITC–anti-Vg5 Abs. More than 70 DETCs from one area were classified as having more than two dendrites (white bars), one or two dendrites (gray bars), or no dendrites (black bars); percentages of each type were calculated. Four independent samples from WT and Ocln2/2 mice were analyzed, and average numbers are shown. (E) DETCs from KikGR and KikGR/Ocln2/2 mice were cultured on epidermal stromal cells for 24 h in the presence of IL-2. They were stimulated for 4 h with biotin–anti-CD3 Abs, by guest on October 2, 2021 followed by allophycocyanin-streptavidin, or were cultured without stimulation. Morphological changes were observed using a FLUOVIEW FV10i confocal microscope (Olympus) at 37˚C and 5% CO2. Rounding cells are indicated by arrows. One representative analysis of three independent experiments is shown. Scale bar, 50 mm. (F) Cells with green fluorescence with spread or rounded shape were counted in 10 mi- croscopic fields at original magnification 340. Total cell numbers (rounded/spread) counted in 10 fields were as follows: WT none: 32/127, WT anti-CD3: 61/ 30, knockout (KO) none: 30/131, KO anti-CD3: 35/ 121. Average percentages of rounding cells are shown. Data are mean 6 SD (n = 10). *p , 0.05, **p , 0.005, ***p , 0.0005.

DNFB-treated epidermis. For this purpose, epidermal sheets were whereas those in wild-type mice had rounded shapes (Fig. 5B, isolated 24 h after DNFB administration and stained with anti- 5C). Next, we investigated the involvement of occludin in gd TCRgd and anti-occludin Abs. Epidermal sheets isolated from T cells during CHS by inducing CHS in Ocln2/2 and wild-type Ocln2/2 mice were also stained as a negative control. We did not mice. After DNFB challenge, ear thickness was much lower in observe occludin staining in Ocln2/2 epidermis. DETCs in Ocln2/2 mice than in wild-type mice. The reduction in CHS in DNFB-treated epidermis were positive for occludin expression, TCRd2/2 mice was more signiﬁcant, indicating that the response whereas those in intact epidermis were not (Fig. 5A). As observed of Ocln2/2 gd T cells to CHS was partially impaired (Fig. 5D). To in UVB-irradiated epidermis, DETCs in Ocln2/2 mice exhibited exclude the possibility that cytokine expression by ab or gd very little morphological change 18 h after DNFB treatment, T cells was impaired in Ocln2/2 mice, we stimulated cells 6 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Migration of gd T cells from skin to the draining lymph nodes is increased after epidermal stress. (A) Schematic diagram of the analysis. (B) FACS profiles of inguinal lymph node cells analyzed 2 d after photoconversion. Cells were obtained from mice without stimulation (n = 2) or from mice subjected to tape stripping (n = 5) or UVB irradiation (n = 2) (upper panel). Photoconverted (KikGR-Red) cells were gated, and expression of TCRgd was analyzed (lower panel). Representative data from three experiments are shown. (C) Numbers of photoconverted cells migrated into the inguinal lymph nodes were calculated from FACS analysis performed at the indicated times after photoconversion. Average numbers from three to five independent tape- stripping experiments are shown (black bars); data are mean 6 SE. Data for UVB irradiation (white bars) and no UVB treatment (gray bars) are averages of two experiments. (D) Numbers of TCRgd+ cells migrating from the skin were calculated from the analysis in (C). (E) Photoconverted cells in inguinal lymph nodes on day 4 were further analyzed using anti-Vg5 Abs. Cells were first gated by forward versus side scatter, and doublet cells were excluded. These size-gated cells were analyzed for KikGR-Green versus KikGR-Red expression. Photoconverted cells (KikGR-Red+) were further analyzed for their expression of TCRgd and Vg5. Representative data from five independent experiments are shown. (F) Numbers of TCRgd+Vg5+ cells migrating from the skin were calculated from the analysis in (C). Data are mean 6 SE. The Journal of Immunology 7

numbers of dendritic cells were found in culture medium of Ocln2/2 samples, indicating that chemotaxis of dendritic cells was not affected by occludin deficiency (Fig. 6B). As we expected, the numbers of gd T cells migrating out of the skin were very low. We also found that significantly fewer gd T cells migrated out of Ocln2/2 skin than wild-type skin. To determine whether dermal gd Tcells express occludin constitutively, we analyzed the expression of occludin in DETCs and dermal gd T cells. DETCs (TCR gd+Vg5+) and dermal gd T cells (TCR gd+Vg52) were sorted from the ear skin of wild-type mice, and relative expression levels of occludin were analyzed by quantitative real-time PCR (Supplemental Fig. 3). Occludin expression in dermal gd T cells was three times higher than that in DETCs in steady-state, suggesting that occludin deficiency also affected the motility of dermal gd T cells. These results suggested that the reduction in CHS responses in Ocln2/2 mice was not due to a defect in dendritic cells but, instead, was a result of the impaired motility of gd T cells.

Discussion DETCs survey epidermal stress–induced molecules and play a Downloaded from pivotal role in maintaining epidermal homeostasis. In this study, we found that DETCs migrated into draining lymph nodes upon activation and promoted immune responses in the skin, indicating that DETCs function in the epidermis, as well as in lymph nodes. Morphological changes occurring upon DETC activation have been documented in wounding (1), allergen administration (7), and li- http://www.jimmunol.org/ gation of NKG2D (2); however, further roles of rounded DETCs are not well understood. At steady-state, dermal gd T cells are mobile, whereas epidermal DETCs remain immobile (24, 25). However, once DETCs are activated in dysregulated epidermis, occludin expression causes them to undergo morphological changes and become motile, ultimately migrating into draining lymph nodes. Occludin is critical for migration of gd IELs (23), which migrate actively within the intraepithelial compartment and lamina propria. by guest on October 2, 2021 This migration is mediated via a homotypic interaction between FIGURE 4. Migration of DETCs to draining lymph nodes is impaired in 2 2 occludin molecules expressed on the surfaces of IELs and epithelial Ocln / mice. (A) KikGR mice, with or without occludin deficiency, were cells. It is possible that occludin is involved in the motility of all gd treated as described in Fig. 3. DETCs were activated by tape stripping, and 2/2 inguinal lymph node cells were analyzed on day 4. Cells were analyzed as T cells. Chemotaxis toward CXCL12 was impaired in Ocln described in Fig. 3E. Representative data from five independent experi- DETCs, indicating that occludin expressed in DETCs, but not in ments are shown. (B) Numbers of DETCs in inguinal lymph nodes derived keratinocytes, is required for DETC motility. In DETCs, occludin from skin on day 4 were calculated from FACS analysis. Few skin-derived expression was induced upon activation, whereas dermal gd Tcells, DETCs were detected in inguinal lymph nodes of Ocln2/2 mice (n = 5). as well as gd IELs, express occludin constitutively. DETCs express (C) Migration of DETCs in vitro was analyzed by Transwell assays. Vg5+ other tight junction molecules, such as ZO-1, as do gd IELs (23); 2/2 cells were isolated by FACS from wild-type (WT) and Ocln DETC therefore, downstream signals of tight junction molecules could 3 6 lines and seeded in the upper chamber of the Transwell (1 10 cells per confer unique features to gd T cells. Fewer dermal gd Tcellsin m 2 2 milliliter in 100 l of medium containing IL-2). Cells that migrated into Ocln / mice compared with wild-type mice migrated out of the the lower chamber, with or without CXCL12, were counted after 4 h. One skin in vitro. However, in vivo analysis using KikGR mice revealed representative assay of three independent experiments is shown. Data are mean 6 SD. *p , 0.05. that the defect in migration from the skin to the draining lymph nodes was observed only in DETCs in Ocln2/2 mice, suggesting that occludin expression in dermal gd T cells may not be involved obtained from skin-draining lymph nodes from wild-type and in their migration into lymph nodes. Ocln2/2 mice with anti-CD3 plus anti-CD28 Abs. Both types of Morphological changes in DETCs after activation were impaired T cells expressed IFN-g and IL-17A at similar levels (Supplemental in Ocln2/2 mice, indicating that occludin-induced cytoskeletal Fig. 2). These results suggested that Ocln2/2 DETCs, which exhibit rearrangement plays a pivotal role in determining cell shape. impairments in cytoskeletal rearrangement and motility after acti- DETCs form immunological synapses at keratinocyte tight junc- vation, failed to migrate and activate other cells involved in CHS. tions; however, reorganization of synapses is induced once these Because the dendritic cells that capture Ags and migrate into the cells are activated in response to keratinocyte stress (16). The draining lymph nodes also play a role in CHS, we analyzed microtubule-organizing center localizes toward immunological chemotaxis of dendritic cells in vitro. For this purpose, ear skin synapses, and the microtubule network is disorganized in from Ocln2/2 and wild-type mice was separated into dorsal and occludin-deficient epithelial cells (26), suggesting that occludin ventral sides and floated dermal side down on medium containing expressed in DETCs plays a role in microtubule-organizing center CCL21. After 48 h, cells migrating out of the skin were collected reorientation and localizes to leading edges of migrating cells. and analyzed by FACS (Fig. 6A). Dendritic cells, as well as gd Signals from CD100 expressed in activated DETCs play a pivotal T cells, migrated out of skin isolated from wild-type mice. Similar role in these morphological changes. In particular, CD100 signals 8 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION

FIGURE 5. Occludin-deﬁcient mice are less sensitive to CHS responses. (A) Wild-type (WT) and Ocln2/2 mice were treated with 0.15% DNFB in acetone/olive oil or with acetone/ olive oil alone (vehicle control) on the ventral Downloaded from side of ears. Mice were sacriﬁced after 24 h, and epidermal sheets were stained with anti-occludin and allophycocyanin-labeled anti-TCRgd Abs, followed by Alexa Fluor 488–conjugated goat anti-rabbit IgG. DETCs expressing occludin (green) and TCRgd (red) appear yellow in the merged image. DETCs expressing both occlu- http://www.jimmunol.org/ din and TCRgd are indicated by arrows. One representative analysis of four independent experiments is shown. Scale bar, 100 mm. (B) Ocln2/2 and WT mice were treated with 0.15% DNFB as in (A). Epidermal sheets were prepared 18 h after treatment and stained with anti- TCRgd. Scale bar, 100 mm. (C) Morphological changes in DETCs were analyzed as in Fig. 2D.

Epidermal sheets were prepared 18 h after by guest on October 2, 2021 DNFB treatment, and DETCs were stained with anti-Vg5 Abs. Data are mean 6 SD. (D)CHS responses were assessed in WT (n =5),Ocln2/2 (n = 7), and TCRd2/2 (n =4)mice.*p , 0.05, **p , 0.005, ***p , 0.0005.

through an interaction with plexin B2 expressed in keratinocytes, T cells, suggesting that these stimuli activated DETCs, as well as leading to changes in morphology (18). However, occludin-deficient dermal gd T cells. Dermal gdT17 cells migrate into draining DETCs expressed CD100 at levels similar to those observed in lymph nodes in imiquimod-induced psoriasis-like dermatitis (27). wild-type DETCs after activation, suggesting that occludin ex- gdT17 cells play an important role in IL-17–mediated inflam- pression may be downstream of CD100 signaling and that CD100- mation; however, the roles of DETCs that normally produce IFN-g deficient DETCs might not induce occludin expression. in draining lymph nodes remain poorly understood. Nielsen et al. Dysregulation of the epidermis induced by tape stripping or (7) demonstrated that DETCs are involved in CHS; DETCs are UVB irradiation increased the migration of Vg5+ and Vg52 gd activated by IL-1b produced in the epidermis and express IL-17. The Journal of Immunology 9

the result of activation of DETCs in response to NKG2D ligand expressed on keratinocytes. In addition to hapten-specific T cells, DETCs are required to transfer CHS responses, and draining lymph node cells on days 4 and 5 following epicutaneous hapten administration can transfer full CHS (30). This observation is in close agreement with our analysis showing that DETCs were detected on days 4 and 5 after epidermal stress, and it seems likely that those DETCs contributed to CHS by helping hapten-specific T cells. As discussed above, gd T cells, but not dendritic cells, exhibited impaired motility in occludin-deficient mice. These results strongly suggest that occludin expressed upon activation plays a pivotal role in DETC function in draining lymph nodes.

Acknowledgments We thank M. Furuse for providing Ocln2/2 mice and Y. Hara for cell sorting.

Disclosures The authors have no ﬁnancial conﬂicts of interest. Downloaded from

FIGURE 6. In vitro chemotaxis of skin dendritic cells is normal in Ocln2/2 mice. (A) Ear skin was prepared from wild-type (WT) and References Ocln2/2 mice and ﬂoated on RPMI 1640 complete medium containing 1. Jameson, J. M., G. Cauvi, D. A. Witherden, and W. L. Havran. 2004. A keratinocyte-responsive gdTCR is necessary for dendritic epidermal T cell 100 ng/ml CCL21. Cells migrating into the medium from the skin were activation by damaged keratinocytes and maintenance in the epidermis. J. collected after 2 d and analyzed by FACS. Cells were gated by forward Immunol. 172: 3573–3579.

versus side scatter, and these size-gated cells were analyzed for TCRgd 2. Strid, J., S. J. Roberts, R. B. Filler, J. M. Lewis, B. Y. Kwong, W. Schpero, http://www.jimmunol.org/ and CD11c expression. One representative analysis of three independent D. H. Kaplan, A. C. Hayday, and M. Girardi. 2008. Acute upregulation of an NKG2D ligand promotes rapid reorganization of a local immune compartment experiments is shown. (B) Average cell numbers from three independent with pleiotropic effects on carcinogenesis. Nat. Immunol. 9: 146–154. assays are shown. Data are mean 6 SD. *p , 0.05. 3. Girardi, M., D. E. Oppenheim, C. R. Steele, J. M. Lewis, E. Glusac, R. Filler, P. Hobby, B. Sutton, R. E. Tigelaar, and A. C. Hayday. 2001. Regulation of cutaneous malignancy by gammadelta T cells. Science 294: 605–609. d2/2 4. Jameson, J., K. Ugarte, N. Chen, P. Yachi, E. Fuchs, R. Boismenu, and Those investigators showed that CHS responses in TCR mice W. L. Havran. 2002. A role for skin gammadelta T cells in wound repair. Science 2/2 were almost equivalent to those in IL-17 mice, suggesting that 296: 747–749. gd T cells expressing IL-17 play a central role in these processes. 5. Sharp, L. L., J. M. Jameson, G. Cauvi, and W. L. Havran. 2005. Dendritic epidermal T cells regulate skin homeostasis through local production of insulin- Occludin deﬁciency did not alter IL-17 expression in gd T cells.

like growth factor 1. Nat. Immunol. 6: 73–79. by guest on October 2, 2021 2 2 CHS responses were reduced in Ocln / mice but not as much as 6. Dieli, F., G. L. Asherson, G. Sireci, R. Dominici, F. Gervasi, S. Vendetti, those in TCRd2/2 mice, suggesting that some gd T cells that V. Colizzi, and A. Salerno. 1997. gd cells involved in contact sensitivity pref- erentially rearrange the Vgamma3 region and require interleukin-7. Eur. J. migrated into draining lymph in an occludin-independent manner Immunol. 27: 206–214. also participated in CHS responses. Alternatively, a subset of re- 7. Nielsen, M. M., P. Lovato, A. S. MacLeod, D. A. Witherden, L. Skov, B. Dyring- Andersen, S. Dabelsteen, A. Woetmann, N. Ødum, W. L. Havran, et al. 2014. sponses may take place in the skin, because DETCs have been IL-1b-dependent activation of dendritic epidermal T cells in contact hypersen- shown to express IL-17 in the epidermis, which plays an important sitivity. J. Immunol. 192: 2975–2983. role in CHS responses (7). 8.Rahimpour,A.,S.R.Mattarollo,M.Yong,G.R.Leggatt,R.J.Steptoe,and I. H. Frazer. 2012. gd T cells augment rejection of skin grafts by enhancing cross- Our experiments in which we tracked cells in the skin by priming of CD8 T cells to skin-derived antigen. J. Invest. Dermatol. 132: 1656–1664. photoconversion clearly demonstrated that DETCs could migrate 9. Shimura, E., N. Hozumi, O. Kanagawa, D. Metzger, P. Chambon, F. Radtke, into draining lymph nodes, and these cells were observed on days 4 S. Hirose, and N. Nakano. 2010. Epidermal gammadelta T cells sense precancerous cellular dysregulation and initiate immune responses. Int. Immunol. 22: 329–340. and 5 after photoconversion. Although we could detect very few 10. Huang, Y., A. Getahun, R. A. Heiser, T. O. Detanico, K. Aviszus, G. A. Kirchenbaum, photoconverted cells in draining lymph nodes, this system was T. L. Casper, C. Huang, M. K. Aydintug, S. R. Carding, et al. 2016. gd T cells shape preimmune peripheral B cell populations. J. Immunol. 196: 217–231. sensitive enough to detect DETCs derived from the epidermis. Cell 11. Heilig, J. S., and S. Tonegawa. 1986. Diversity of murine g genes and expression traffic from the skin to the draining lymph nodes, including gd in fetal and adult T lymphocytes. Nature 322: 836–840. T cells and dendritic cells, was increased by epidermal stress, 12. Barbee, S. D., M. J. Woodward, G. Turchinovich, J. J. Mention, J. M. Lewis, L. M. Boyden, R. P. Lifton, R. Tigelaar, and A. C. Hayday. 2011. Skint-1 is a indicating that gd T cells are innate immune cells that contribute highly specific, unique selecting component for epidermal T cells. Proc. Natl. to adaptive immunity. Migration of dendritic cells is regulated by Acad. Sci. USA 108: 3330–3335. CCR7, which is upregulated by activation, but the mechanisms by 13. Boyden, L. M., J. M. Lewis, S. D. Barbee, A. Bas, M. Girardi, A. C. Hayday, R. E. Tigelaar, and R. P. Lifton. 2008. Skint1, the prototype of a newly identified which gd T cells migrate from the skin to draining lymph nodes immunoglobulin superfamily gene cluster, positively selects epidermal gam- are not well understood (28). The reduction in the number of gd madelta T cells. Nat. Genet. 40: 656–662. 14. Lewis, J. M., M. Girardi, S. J. Roberts, S. D. Barbee, A. C. Hayday, and T cells in draining lymph nodes, and especially the lack of DETCs R. E. Tigelaar. 2006. Selection of the cutaneous intraepithelial gammadelta+ 2/2 in Ocln mice, demonstrated that DETCs use occludin for cy- T cell repertoire by a thymic stromal determinant. Nat. Immunol. 7: 843–850. toskeletal changes and movement. DETCs became motile only 15. Turchinovich, G., and A. C. Hayday. 2011. Skint-1 identifies a common molecular mechanism for the development of interferon-g-secreting versus after occludin expression following activation. interleukin-17-secreting gd T cells. Immunity 35: 59–68. CHS is a T cell–mediated inflammatory response induced by 16. Chodaczek, G., V. Papanna, M. A. Zal, and T. Zal. 2012. Body-barrier surveil- skin allergens, and IL-1b–mediated DETC activation plays an lance by epidermal gd TCRs. Nat. Immunol. 13: 272–282. 17. Witherden, D. A., P. Verdino, S. E. Rieder, O. Garijo, R. E. Mills, L. Teyton, important role in CHS pathogenesis (7). Recent work showed that W. H. Fischer, I. A. Wilson, and W. L. Havran. 2010. The junctional adhesion activation of DETCs can be induced by NKG2D ligands, which molecule JAML is a costimulatory receptor for epithelial gammadelta T cell activation. Science 329: 1205–1210. are upregulated on keratinocytes by allergens (29). Occludin ex- 18. Witherden, D. A., M. Watanabe, O. Garijo, S. E. Rieder, G. Sarkisyan, pression was observed in DNFB-treated epidermis, potentially as S. J. Cronin, P. Verdino, I. A. Wilson, A. Kumanogoh, H. Kikutani, et al. 2012. 10 OCCLUDIN-DEPENDENT MIGRATION OF DETCs UPON ACTIVATION

The CD100 receptor interacts with its plexin B2 ligand to regulate epidermal gd 25. Gray, E. E., K. Suzuki, and J. G. Cyster. 2011. Cutting edge: identification of a T cell function. Immunity 37: 314–325. motile IL-17-producing gammadelta T cell population in the dermis. J. Immunol. 19. Saitou, M., M. Furuse, H. Sasaki, J.-D. Schulzke, M. Fromm, H. Takano, 186: 6091–6095. T. Noda, and S. Tsukita. 2000. Complex phenotype of mice lacking occludin, a 26. Du, D., F. Xu, L. Yu, C. Zhang, X. Lu, H. Yuan, Q. Huang, F. Zhang, H. Bao, component of tight junction strands. Mol. Biol. Cell 11: 4131–4142. L. Jia, et al. 2010. The tight junction protein, occludin, regulates the directional 20. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A. R. Clarke, migration of epithelial cells. Dev. Cell 18: 52–63. M. L. Hooper, A. Farr, and S. Tonegawa. 1993. T cell receptor d gene mutant 27. Gray, E. E., F. Ramı´rez-Valle, Y. Xu, S. Wu, Z. Wu, K. E. Karjalainen, and mice: independent generation of ab T cells and programmed rearrangements of J. G. Cyster. 2013. Deficiency in IL-17-committed Vg4(+) gd T cells in a gd TCR genes. Cell 72: 337–348. spontaneous Sox13-mutant CD45.1(+) congenic mouse substrain provides pro- 21. Tomura, M., A. Hata, S. Matsuoka, F. H. Shand, Y. Nakanishi, R. Ikebuchi, tection from dermatitis. Nat. Immunol. 14: 584–592. S. Ueha, H. Tsutsui, K. Inaba, K. Matsushima, et al. 2014. Tracking and 28. Nakamizo, S., G. Egawa, M. Tomura, S. Sakai, S. Tsuchiya, A. Kitoh, T. Honda, quantification of dendritic cell migration and antigen trafficking between the skin A. Otsuka, S. Nakajima, T. Dainichi, et al. 2015. Dermal Vg4(+) gd T cells and lymph nodes. Sci. Rep. 4: 6030. possess a migratory potency to the draining lymph nodes and modulate CD8(+) 22. Havran, W. L., and J. M. Jameson. 2010. Epidermal T cells and wound healing. T-cell activity through TNF-a production. J. Invest. Dermatol. 135: 1007–1015. J. Immunol. 184: 5423–5428. 29. Nielsen, M. M., B. Dyring-Andersen, J. D. Schmidt, D. Witherden, P. Lovato, 23. Edelblum, K. L., L. Shen, C. R. Weber, A. M. Marchiando, B. S. Clay, Y. Wang, A. Woetmann, N. Ødum, S. S. Poulsen, W. L. Havran, C. Geisler, and I. Prinz, B. Malissen, A. I. Sperling, and J. R. Turner. 2012. Dynamic migration C. M. Bonefeld. 2015. NKG2D-dependent activation of dendritic epidermal of gd intraepithelial lymphocytes requires occludin. Proc. Natl. Acad. Sci. USA T cells in contact hypersensitivity. J. Invest. Dermatol. 135: 1311–1319. 109: 7097–7102. 30. Dieli, F., W. Ptak, G. Sireci, G. C. Romano, M. Potestio, A. Salerno, and 24. Hayday, A. C. 2009. Gammadelta T cells and the lymphoid stress-surveillance G. L. Asherson. 1998. Cross-talk between Vb8+ and gd+ T lymphocytes in response. Immunity 31: 184–196. contact sensitivity. Immunology 93: 469–477. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021